Therapeutic Use of CD31 Expressing Cells

ABSTRACT

As described below, the present invention features compositions and methods related to the isolation, culture and therapeutic use of CD31-expressing cells.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/854,957, filed Oct. 27, 2006 and U.S. Provisional Application No.60/855,998, filed Oct. 31, 2006, the contents of which are incorporatedherein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grants from the NationalInstitutes of Health, Grant No. 1RO1 HL 079137. The government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

Despite many breakthroughs in cardiovascular medicine, the treatment ofischemic cardiovascular diseases remains among the most prominent healthchallenges worldwide. The identification of adult stem or progenitorcells capable of contributing to tissue regeneration has raised thepossibility that cell therapy could be employed for repair of ischemicdamaged tissues. Current investigations have suggested bone marrow (BM)cells as a potential source for adult stem or progenitor cells.BM-derived stem cells appear to have the capacity to repopulate manynonhematopoietic tissues, such as vessel and muscle. The promisingresults from initial experimental studies on BM-derived stem cells havealready promoted the initiation of clinical trials for the treatment ofacute myocardial infarction, ischemic cardiomyopathy, and limb ischemia.Yet knowledge relating to adult stem cells populations is incomplete.One of the most important and unresolved issues in cell therapy is theselection of ideal cells for regeneration. Even though various kinds ofstem or progenitor cells have been proposed and shown to be effectivefor cardiovascular regeneration, each cell type has its own pitfalls.For example, un-fractionated whole BM cells may encounter unexpected andpotentially serious adverse effects, such as intramyocardialcalcification. CD34⁺ cells or c-kit⁺ cells exist in low numbers in BM(less than 1%), therefore, the mobilization process is required toobtain sufficient numbers of cells to be used for cell therapy.Endothelial progenitor cells or mesenchymal stem cells are cultureexpandable cells, that require large amounts of serum for culture andneed from days to months to be prepared for clinical use. Better methodsfor selecting, isolating, and culturing stem cells for use inregenerative medicine are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods related to the isolation, culture and therapeutic use ofCD31-expressing cells.

In one aspect, the invention generally features a method of repairing orregenerating a tissue in a subject in need thereof. The method involvescontacting the tissue with a CD31⁺ cell, thereby repairing orregenerating the tissue.

In another aspect, the invention provides a method for increasingangiogenesis in a subject in need thereof. The method involvescontacting the tissue with a CD31⁺ cell, thereby repairing orregenerating the tissue.

In another aspect, the invention features a method for amelioratingischemia-related tissue damage in a subject in need thereof, the methodinvolves administering to the subject a CD31⁺ cell having the potentialto differentiate into an endothelial cell; increasing angiogenesis andincreasing secretion of a paracrine factor or cytokine in a tissue ofthe subject, thereby ameliorating ischemia in the subject. In variousembodiments, the ischemia-related tissue damage is associated with heartfailure, myocardial infarction, limb ischemia, stroke, transientischemia, or reperfusion injury.

In yet another aspect, the invention features a method for amelioratinga neuropathy in a subject in need thereof. The method involvesadministering to the subject a CD31⁺ cell having the potential todifferentiate into an endothelial cell; and increasing angiogenesis in aneural tissue of the subject, thereby ameliorating a neuropathy in thesubject.

In yet another aspect, the invention features a method for amelioratingheart failure in a subject in need thereof. The method involvesadministering to a cardiac tissue a CD31⁺ cell; and increasingangiogenesis in the cardiac tissue, thereby ameliorating heart failurein the subject.

In yet another aspect, the invention features a method for amelioratingliver or renal failure in a subject in need thereof. The method involvesadministering to a liver or renal tissue a CD31⁺ cell; and engraftingthe CD31⁺ cell into the liver or renal tissue, thereby amelioratingliver or renal failure in the subject. In various embodiments of theabove aspects, the cell is locally or systemically administered. Inother embodiments of the above aspects, the cell is integrated into thetissue.

In yet another aspect, the invention features a method for identifying amultipotent stem cell. The method involves identifying a cell thatexpresses CD31⁺, and isolating the cell. In one embodiment, theidentification step involves an immunoassay.

In yet another aspect, the invention features a packaged pharmaceuticalcontaining a therapeutically effective amount of a CD31⁺ cell, andinstructions for use in treating a subject having a conditioncharacterized by excess cell death. In one embodiment, the compositionfurther contains a therapeutic polypeptide.

In yet another aspect, the invention features a packaged pharmaceuticalcontaining a therapeutically effective amount of a CD31⁺ cell having thepotential to differentiate into an endothelial cell, and instructionsfor use in treating or preventing a ischemic disease in a subject. Inone embodiment, the cell is genetically modified.

In yet another aspect, the invention features a method for identifyingan agent useful for enhancing the transdifferentiation of a CD31⁺ cell,the method involving contacting a CD31⁺ cell with an agent; andmeasuring an increase in the expression of a protein not expressed in anuntreated CD31⁺ control cell, where an increase in protein expression inthe treated cell, as compared to the untreated cell identifies the agentas useful for transdifferentiating the CD31⁺ cell. In one embodiment,the protein is insulin. In another embodiment, the protein is anendothelial cell marker, a liver cell marker, or a renal cell marker.

In yet another aspect, the invention features a method for culturing aCD31-expressing multipotent stem cell.

In another aspect, the invention provides a method for amelioratingischemia related tissue damage in a subject in need thereof, the methodinvolving administering to the subject a CD31⁺ cell; and increasingsecretion of a paracrine factor or cytokine in a tissue of the subject,thereby ameliorating ischemia related tissue damage in the subject.

In yet another aspect, the invention provides a method for amelioratingheart failure in a subject in need thereof, the method involvingadministering to a cardiac tissue a CD31⁺ cell; and increasing secretionof a paracrine factor or cytokine in the cardiac tissue, therebyameliorating heart failure in the subject.

In yet another aspect, the invention provides a method for increasingwound healing in a tissue of subject in need thereof, the methodcomprising: administering to said tissue a CD31⁺ cell thereby increasingwound healing.

In still another aspect, the invention provides a method for increasingwound healing in a tissue of a subject in need thereof, the methodinvolving administering to said tissue a CD31⁺ cell; and increasingangiogenesis thereby increasing wound healing.

In still another aspect, the invention provides a method for increasingwound healing in a tissue of a subject in need thereof, the methodinvolving administering to said tissue a CD31⁺ cell; and increasingsecretion of a paracrine factor or cytokine in said tissue, therebyincreasing wound healing in the subject.

In still another aspect, the invention provides a method for increasingwound healing in a tissue of a subject in need thereof, the methodinvolving administering to said tissue a CD31⁺ cell; and engrafting theCD31⁺ cell into the tissue, thereby increasing wound healing.

In yet another aspect, the invention provides a method for treating ahematologic disease in a subject in need thereof, the method involvingadministering to said tissue a CD31+Lin− cell thereby treating saidhematologic disease (e.g., leukemia, lymphoma, myelodysplastic syndrome,pancytopenia, anemia, thrombocytopenia, leucopenia).

In yet another aspect, the invention provides a method for identifying amultipotent stem cell, the method comprising: identifying a cell thatexpresses CD31⁺.

In another aspect, the invention provides a method for identifying amultipotent stem cell, the method involving identifying a cell thatexpresses CD31⁺ and does not express Lin.

In yet another aspect, the invention provides a method of isolating amultipotent stem cell the method involving isolating a CD31⁺ cell; andselecting said CD31⁺ cell. In another aspect, the invention provides amethod of isolating a multipotent stem cell the method involvingisolating a CD31+lin− cell; and selecting said CD31+lin− cell.

In yet another aspect, the invention provides a method for culturing aCD31+ cell involving isolating stem cells from bone marrow, peripheralblood or umbilical cord blood; identifying a CD31⁺ cell; and expandingsaid CD31⁺ cell.

In another aspect, the invention provides a method for culturing aCD31⁺lin⁻ cell involving isolating stem cells from bone marrow,peripheral blood or umbilical cord blood; identifying a CD31⁺lin⁻ cell;and expanding said CD31⁺lin⁻ cell.

In another aspect, the invention provides a method of repairing orregenerating a tissue in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; and administering saidCD31⁺ cell to said subject thereby repairing or regenerating saidtissue.

In still another aspect, the invention provides a method for increasingangiogenesis in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; and administering saidCD31⁺ cell to said subject thereby increasing angiogenesis.

In still another aspect, the invention provides a method of amelioratingischemia related tissue damage in a subject in need thereof, the methodinvolving isolating a CD31⁺ cell from bone marrow, peripheral blood orcord blood; expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells;administering said CD31⁺ cell to said subject; and increasingangiogenesis in a tissue of said subject, thereby ameliorating ischemiain said subject.

In still another aspect, the invention provides a method of amelioratingischemia related tissue damage in a subject in need thereof, the methodinvolving isolating a CD31⁺ cell from bone marrow, peripheral blood orcord blood; expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells;administering said CD31⁺ cell to said subject; and increasing secretionof a paracrine factor or cytokine in a tissue of said subject, therebyameliorating ischemia in said subject.

In still another aspect, the invention provides a method of amelioratinga neuropathy in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; administering saidCD31⁺ cell to said subject; and increasing angiogenesis in a neuraltissue of said subject, thereby ameliorating a neuropathy in saidsubject.

In still another aspect, the invention provides a method of amelioratinga neuropathy in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; administering saidCD31⁺ cell to said subject; and increasing secretion of a paracrinefactor or a cytokine in a neural tissue of said subject, therebyameliorating a neuropathy in said subject.

In still another aspect, the invention provides a method of amelioratingheart failure in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; administering saidCD31⁺ cell to cardiac tissue of said subject; and increasingangiogenesis in said cardiac tissue, thereby ameliorating heart failurein the subject.

In another aspect, the invention provides a method of ameliorating heartfailure in a subject in need thereof, the method involving isolating aCD31⁺ cell from bone marrow, peripheral blood or cord blood; expandingsaid CD31⁺ cell in vitro to obtain a cell population enriched in bonemarrow-derived stem or progenitor cells; administering said CD31⁺ cellto cardiac tissue of said subject; and increasing secretion of aparacrine factor or cytokine in said cardiac tissue, therebyameliorating heart failure in the subject.

In another aspect, the invention provides a method of ameliorating heartfailure in a subject in need thereof, the method involving isolating aCD31⁺ cell from bone marrow, peripheral blood or cord blood; expandingsaid CD31⁺ cell in vitro to obtain a cell population enriched in bonemarrow-derived stem or progenitor cells; administering said CD31⁺ cellto cardiac tissue of said subject; and increasing myogenesis in saidcardiac tissue, thereby ameliorating heart failure in the subject.

In another aspect, the invention provides a method of ameliorating liveror renal failure in a subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; administering saidCD31⁺ cell to the liver or renal tissue of said subject; and engraftingthe CD31⁺ cell into the liver or renal tissue, thereby amelioratingliver or renal failure in the subject.

In another aspect, the invention provides a method for increasing woundhealing in a tissue of subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; and administering tosaid tissue a CD31⁺ cell thereby increasing wound healing.

In another aspect, the invention provides a method for increasing woundhealing in a tissue of subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; administering to saidtissue a CD31⁺ cell thereby increasing angiogenesis thereby increasingwound healing.

In another aspect, the invention provides a method for increasing woundhealing in a tissue of subject in need thereof, the method involvingisolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; and administering tosaid tissue a CD31⁺ cell thereby increasing secretion of a paracrinefactor or cytokine in said tissue, thereby increasing wound healing inthe subject.

In yet another aspect, the invention provides a method for increasingwound healing in a tissue of a subject in need thereof, the methodinvolving isolating a CD31⁺ cell from bone marrow, peripheral blood orcord blood; expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells; andadministering to said tissue a CD31⁺ cell; and engrafting the CD31+ cellinto the tissue, thereby increasing wound healing.

In yet another aspect, the invention provides a method for treating ahematologic disease in a tissue of a subject in need thereof, the methodinvolving isolating a CD31⁺lin− cell from bone marrow, peripheral bloodor cord blood; expanding said CD31⁺lin− cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells; andadministering to said tissue a CD31⁺lin− cell; and thereby treating saidhematologic disease.

In yet another aspect, the invention provides an isolated CD31⁺, lin⁻cell or a pharmaceutical composition comprising and isolated CD31⁺Lin⁻cell, for example, an isolated CD31⁺Lin− cell obtained by the method ofa previous aspect.

In various embodiments of the above aspects, the tissue is a muscletissue, cardiac tissue, neural tissue, liver tissue, pancreatic tissue,bone tissue, cartilage, renal tissue, or a tissue characterized byexcess cell death. In other embodiments of any of the above aspects, thesubject has or has a propensity to develop myocardial infarction,congestive heart failure, peripheral vascular obstructive disease,ischemia, limb ischemia, stroke, transient ischemia, reperfusion injury,peripheral neuropathy, toxic neuropathy, diabetic dementia, or autonomicneuropathy, liver failure, renal failure, diabetes, rheumatoidarthritis, osteoarthritis, and osteoporosis. In other embodiments of theabove aspects, the cell is integrated into the vasculature of thetissue. In still other embodiments of the above aspects, the CD31⁺ cellis isolated and expanded in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells prior to beingadministered to the host subject. In still other embodiments of theabove aspects, the cell is genetically modified. In still otherembodiments of the above aspects, the cell is an endothelial progenitorcell (EPC). In other embodiments of the above aspects, the cell isisolated from the bone marrow of a donor subject. If desired, the donorand the subject receiving the cell are the same individual. In otherembodiments of the above aspects, the cell is a human multipotent stemcell that expresses a normal, increased, or reduced levels of a markerselected from any one or more of: CD90, CD117, CD34, CD113, FLK-1,tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class Ireceptor and MHC class II receptor, as compared to a CD31− cell. Instill other embodiments, the cell expresses or expresses altered levelsof at least two, three, four, or all markers. In still otherembodiments, the method of the invention further involves administeringto the subject a therapeutic polypeptide or a nucleic acid encoding atherapeutic polypeptide. In still other embodiments, the cell is locallyor systemically administered. In various embodiments of any of the aboveaspects, the subject has or has a propensity to develop any one or moreof myocardial infarction, congestive heart failure, peripheral vascularobstructive disease, ischemia, limb ischemia, stroke, transientischemia, reperfusion injury, peripheral neuropathy, diabeticneuropathy, toxic neuropathy, diabetic dementia, or autonomicneuropathy, spinal cord injury, leukemia, lymphoma, myelodysplasticsyndrome, pancytopenia, anemia, thrombocytopenia, leukopenia, liverfailure, renal failure, diabetes, rheumatoid arthritis, osteoarthritis,skin wound, diabetic foot or ulcer, gangrene, diabetic wound andosteoporosis. In various embodiments of the above aspects, the CD31+cell is lineage depleted; is isolated from bone marrow, peripheral bloodor umbilical cord blood of a donor subject (e.g., a mammal, such as ahuman). In various embodiments of the above aspects, the cell expressesat least one of Sca-1 or c-kit; does not express Lin. In variousembodiments of the above aspects, the identification step involves animmunoassay. In various embodiments of the above aspects, the protein isa cardiomyogenic marker or neural marker.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DEFINITIONS

By “platelet/endothelial cell adhesion molecule (CD31) polypeptide” ismeant a protein or fragment thereof having at least 85% identity to theamino acid sequence provided at NP_(—)000433 that acts as a marker forcells having hemangioblastic activity.

By “CD31 nucleic acid sequence” is meant a polynucleotide encoding aCD31 protein.

By “CD31⁺ cell” is meant a cell that expresses a detectable level ofCD31 polypeptide, polynucleotide, or fragment thereof. A CD31⁺ cell ofthe invention has hemangioblastic activity. A CD31⁺ cell according tothe invention includes cells that have the potential to give rise toangioblasts or hematopoietic cells. A CD31⁺ cell according to theinvention also includes cells that have the potential to differentiateinto endothelial cells. In one embodiment, a CD31⁺ cell expresses anormal level or an altered (increased or decreased) level as compared toa CD31⁻ cell of at least one of the following markers: CD90, CD117,CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117,CD133, MHC class I receptor and MHC class II receptor. In anotherembodiment, a CD31⁺ cell expresses at least one of Sca-1 or c-Kit. Inanother embodiment, a CD31⁺ cell is Lin−. The invention encompassesCD31⁺ cells derived from sources including but not limited to peripheralblood, including umbilical cord blood, for example human umbilical cordblood, bone marrow and hematopoietic stem cells. The invention alsoencompasses CD31⁺ cells from mammals and, in particular, humans.

By “stem cell” is meant an undifferentiated cell which is capable ofessentially unlimited propagation either in vivo or ex vivo and capableof differentiation to other cell types. This can be to certaindifferentiated, committed, immature, progenitor, or mature cell typespresent in the tissue from which it was isolated, or dramaticallydifferentiated cell types, such as for example the erythrocytes andlymphocytes that derive from a common precursor cell, or even to celltypes at any stage in a tissue completely different from the tissue fromwhich the stem cell is obtained. For example, blood stem cells maybecome brain cells or liver cells, neural stem cells can become bloodcells, such that stem cells are pluripotential, and given theappropriate signals from their environment, they can differentiate intoany tissue in the body.

By “hemangioblastic activity” is meant having the potential to give riseto angioblasts, endothelial cells and hematopoietic cells. By “havingthe potential to give rise to angioblasts and hematopoietic cells” ismeant having the ability to produce one or more cells having anangioblastic and/or hematopoietic phenotype under the appropriate invitro or in vivo culture or implantation conditions.

By “angioblast” is meant a cell derived from a hemangioblast from whichblood vessel growth originates during angiogenesis or vasculogenesis. Anangioblast can be an endothelial cell precursor. In one embodiment, an“angioblast” expresses at least one of Flk-1 or CD34.

By “hematopoietic cell” is meant a stem cell which gives rise to all theblood cell types including myeloid (monocytes and macrophages,neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells) and lymphoid lineages(T-cells, B-cells, NK-cells). The hematopoietic tissue contains cellswith long term and short term regeneration capacities and committedmultipotent, oligopotent and unipotent progenitors.

By “transdifferentiation” is meant an alteration in a cell such that thetransdifferentiated cell expresses a detectable level of at least oneprotein of interest not typically expressed in the cell.

By “a cell having the potential to differentiate into an endothelialcell” is meant any cell that can when cultured or implanted undersuitable conditions give rise to cells having an endothelial cellphenotype, expressing one or more endothelial cell markers, or having anendothelial cell function. The term “a cell having the potential todifferentiate into an endothelial cell” includes but is not limited tomultipotent stem cells, endothelial progenitor cells, mesenchymal stemcells, mononuclear cells, or progenitors or progeny thereof. In oneembodiment, a “cell having the potential to differentiate into anendothelial cell” expresses at least one of a cytokine including but notlimited to VEGF, IGF-1 and bFGF.

A “cell having the potential to differentiate into an endothelial cell”differentiates into an endothelial cell that expresses one or moreendothelial cell markers including but not limited to KDR, endothelialnitric oxidase synthase, VE-Cadherin, CD34, FLK-1, Tie2, CD31,VonWillebrand Factor, CD136 or Factor 8.

An “endothelial cell” according to the invention may perform anendothelial cell function including but not limited to uptake ofDiI-acetylated low-density lipoprotein (DiI-acLDL) and binding oflectin.

By “repair” as it refers to a tissue or organ is meant ameliorate damageor disease in a tissue or organ.

By “regenerate” is meant capable of contributing at least one cell tothe repair of de novo construction of a tissue or organ.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “decrease” is meant any negative change. Exemplary decreases include5%, 10%, 20%, 25%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or even by asmuch as 100% compared to a control. Exemplary decreases also include atleast 1-fold (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more).

By “angiogenesis” is meant the growth of new blood vessels. Such growthmay originate from an existing blood vessel or by the development of newblood vessels originating from stem cells, angioblasts, or otherprecursor cells. These stem cells can be recruited from bone marrowendogenously or implanted therapeutically. Methods for measuringangiogenesis are standard, and are described, for example, in Jain etal. (Nat. Rev. Cancer 2: 266-276, 2002). Angiogenesis can be assayed bymeasuring the number of non-branching blood vessel segments (number ofsegments per unit area), the functional vascular density (total lengthof perfused blood vessel per unit area), the vessel diameter, or thevessel volume density (total of calculated blood vessel volume based onlength and diameter of each segment per unit area). Methods formeasuring angiogenesis are standard in the art and are described, forexample, in Jain et al., (Nat. Rev. Cancer 2: 266-276, 2002).

By “derived from” is meant the process of obtaining a progeny cell.“Derived from” also means obtained from a specified source.

By “engraft” is meant the process of cellular contact and incorporationinto an existing tissue of interest (e.g., a blood vessel ormicrovasculature) in vivo.

By “genetically modified” is meant comprising a heterologouspolynucleotide, such as an expression vector.

By “increase in angiogenesis” is meant a positive change in blood vesselformation as measured by standard assays, such as those describedherein. Desirably, an agent that modulates blood vessel formation willincrease blood vessel formation (e.g., angiogenesis, vasculogenesis,formation of an immature blood vessel network, blood vessel remodeling,blood vessel stabilization, blood vessel maturation, blood vesseldifferentiation, or establishment of a functional blood vessel network)in a neural tissue or organ or microvascular scaffold.

By “increase” is meant any positive change. Exemplary increases include5%, 10%, 20%, 25%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or even by asmuch as 100%, 150%, or 200% compared to a control. Exemplary increasesalso include at least 1-fold (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more).

By “integrated” is meant incorporated into a tissue.

A method of “administration” useful according to the invention includesbut is not limited to topical application, intravenous drip orinjection, subcutaneous, intramuscular, intraperitoneal, intracranialand spinal injection, ingestion via the oral route, inhalation,trans-epithelial diffusion (such as via a drug-impregnated, adhesivepatch) or by the use of an implantable, time-release drug deliverydevice, which may comprise a reservoir of exogenously-produced agent ormay, instead, comprise cells that produce and secrete the therapeuticagent. Additional methods of administration are provided hereinbelow.

By “locally administered” is meant provided to a cell, extracellularspace, tissue, organ, or circulatory vessel supplying such a cell,tissue, or organ, under conditions suitable to achieve a therapeuticeffect. Typically, a cell of the invention that is “locallyadministered” is injected into the tissue or nearby tissue which is inneed of CD31⁺ cells for treatment, a muscle tissue comprising a neuronunder conditions that provide for an increase in angiogenesis orvascularity in the neuron.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity as compared to an appropriate control orreference, that is associated with a disease or disorder.

By “pluripotent stem cell” or “multipotent stem cell” is meant a cellhaving the potential to differentiate into more than one cell type.Exemplary cell types include, but are not limited to, endothelial cells,smooth muscle cells, and muscle cells.

By “paracrine”, as it refers to secretion, is meant secretion of variousbiological factors. Biological factors subject to secretion include butare not limited to cytokines and growth factors.

By “neuropathy” is meant any pathology that disrupts neural function.

By “neural function” is meant any function of the nervous system, e.g.neural signaling, neural conductance, sensorimotor function or cognitivefunction.

By “reference” is meant a standard or control condition. A “controlsubject” means a subject that is not diagnosed with, is not suspected ofhaving, and does not have a propensity to develop a disease of interest.A “control tissue” means a tissue derived from a control subject.

By “positioned for expression” is meant that the polynucleotide of theinvention (e.g., a DNA molecule) is positioned adjacent to a DNAsequence which directs transcription and, for proteins, translation ofthe sequence (i.e., facilitates the production of, for example, arecombinant polypeptide of the invention, or an RNA molecule).

By “propensity” is meant at risk for developing pathology. Such risk canbe genetic, environmental, or behavioral.

By “expansion” is meant the propagation of a cell or cells prior to orfollowing terminal differentiation. A cell is “expanded” when it ispropagated in culture and gives rise by cell division to other cellsand/or progenitor cells. Expansion of cells may occur spontaneously ascells proliferate in a culture or it may require certain growthconditions, such as a minimum cell density, cell confluence on theculture vessel surface, or the addition of chemical factors such asgrowth factors, differentiation factors, or signaling factors.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “isolation phenotype” is meant the structural and/or functionalcharacteristics of a stem cell upon isolation.

By “expansion phenotype” is meant the structural and/or functionalcharacteristics of a stem cell during or following expansion. Theexpansion phenotype can be identical to the isolation phenotype, oralternatively, the expansion phenotype can be more differentiated thanthe isolation phenotype. In one embodiment, the expansion or isolationphenotype is characterized by an alteration in the expression of amarker.

By “differentiation” is meant the developmental process of commitment toa particular cell fate. Differentiation to a particular cell fatetypically includes the acquisition of characteristic markers,phenotypes, or functions (e.g., endothelial cell markers or functions).

By “isolated” is meant separated from the molecular and/or cellularcomponents that naturally accompany the cell, polypeptide, orpolynucleotide. “Isolating” a cell refers to the process of removing acell from a sample and separating away other cells which are not thedesired cell type. An isolated cell will be generally free fromcontamination by other cell types. Isolated cells will generally be atleast 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 98% or 99% pure. Thepurity of isolated cells can also be about 50 to about 55%, about 55 toabout 60%, and about 65 to about 70%. More desirably, the purity isabout 70 to about 75%, about 75 to about 80%, about 80 to about 85%; andstill more desirably the purity is about 85 to about 90%, about 90 toabout 95%, and about 95 to about 100%.

By “mesenchymal stem cell” is meant a cell derived from the mesodermallayer that is pluripotent and can develop into a connective orsupporting tissue, smooth muscle, vascular endothelium, or blood cells.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to decreasing theprobability of developing a disorder or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disorder orcondition.

By “progenitor cell” is meant a multipotent stem cell that is capable ofgenerating (e.g., by differentiation or division) a differentiated cell.An endothelial progenitor cell that is capable of generating anendothelial cell may express this capability when grown underappropriate in vitro or in vivo conditions, such as those describedherein.

By “stem cell” is meant a cell capable of giving rise to one or morecell types.

By “differentiated cell” is meant a cell that expresses one or moremarkers characteristic of a particular differentiated cell type orexhibits at least one biological function associated with adifferentiated cell type.

By “progeny” is meant a cell derived from a multipotent stem cell of theinvention. Progeny include without limitation progenitor cells,differentiated cells, and terminally differentiated cells.

By “tissue” is meant a collection of cells having a similar morphologyand function.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated. Additionally, by“treating” is meant preventing disease (for example a disease includingbut not limited to myocardial infarction, congestive heart failure,peripheral vascular obstructive disease, ischemia, limb ischemia,stroke, transient ischemia, reperfusion injury, peripheral neuropathy,toxic neuropathy, diabetic dementia, or autonomic neuropathy, liverfailure, renal failure, diabetes, rheumatoid arthritis, osteoarthritis,and osteoporosis in a subject at risk thereof.

As used herein, “diagnosis” refers to a process of determining if anindividual is afflicted with a disease or ailment, for examplemyocardial infarction, congestive heart failure, peripheral vascularobstructive disease, ischemia, limb ischemia, stroke, transientischemia, reperfusion injury, peripheral neuropathy, toxic neuropathy,diabetic dementia, or autonomic neuropathy, liver failure, renalfailure, diabetes, rheumatoid arthritis, osteoarthritis, andosteoporosis.

By “therapeutic polypeptide” means a protein or analog thereof that hasthe potential of positively affecting the function of an organism. Atherapeutic polypeptide may decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of disease,disorder, in an organism. In various embodiments, therapeuticpolypeptides (e.g., angiogenic factors, neurotrophic factors,pleiotrophic factors) support neuronal or endothelial cell survival,growth, or proliferation.

By “therapeutically effective amount” is meant the amount required toameliorate the symptoms of a disease relative to an untreated patient.The effective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a neuropathy varies dependingupon the manner of administration, the age, body weight, and generalhealth of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

By “agent” is meant a polypeptide, polynucleotide or small compound.Polypeptide agents include growth factors, cytokines, hormones or smallmolecules, or to genetically-encoded products that modulate cellfunction (e.g., induce cell fate, increase expansion, inhibit or promotecell growth and survival). For example, “expansion agents” are agentsthat increase proliferation and/or survival of stem cells.“Differentiation agents” are agents that induce differentiation intocommitted cell lineages.

By “subject” is meant any warm-blooded animal including but not limitedto a human, cow, horse, pig, sheep, goat, bird, mouse, rat, dog, cat,monkey, baboon, or the like.

By “subject in need” is meant a warm-blooded animal that is diagnosedwith a disease, for example including but not limited to myocardialinfarction, congestive heart failure, peripheral vascular obstructivedisease, ischemia, limb ischemia, stroke, transient ischemia,reperfusion injury, peripheral neuropathy, toxic neuropathy, diabeticdementia, or autonomic neuropathy, liver failure, renal failure,diabetes, rheumatoid arthritis, osteoarthritis, and osteoporosis.

A “subject in need” also means a warm-blooded animal that is suspectedof having a disease including but not limited to myocardial infarction,congestive heart failure, peripheral vascular obstructive disease,ischemia, limb ischemia, stroke, transient ischemia, reperfusion injury,peripheral neuropathy, toxic neuropathy, diabetic dementia, or autonomicneuropathy, liver failure, renal failure, diabetes, rheumatoidarthritis, osteoarthritis, and osteoporosis.

The term “obtaining” as in “obtaining the agent” is intended to includepurchasing, synthesizing or otherwise acquiring the agent (or indicatedsubstance or material).

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

By “octamer-binding transcription factor 4 (Oct4) polypeptide” is meanta transcription factor having at least 85% identity to GenBank AccessionNo. NP_(—)976034 or NP_(—)002692 that regulates tissue-specific geneexpression.

By “octamer-binding transcription factor 4 (Oct4) nucleic acid sequence”is meant a polynucleotide encoding a Oct4 polypeptide.

By “RNA exonuclease 4 (Rex4) polypeptide” is meant a polypeptide havingat least 85% identity to NP_(—)065118 that functions in mitosis.

By “RNA exonuclease 4 (Rex4) nucleic acid sequence” is meant apolynucleotide encoding a Rex4 polypeptide.

By “nanog polypeptide” is meant a polypeptide having at least 85%homology to NP_(—)079141 that functions in maintaining the pluripotencyof a nanog expressing cells.

By “nanog nucleic acid sequence” is meant a polynucleotide encoding ananog protein.

By “sex-determining region Y-box 2 polypeptide (Sox2)” is meant atranscription factor having at least 85% identity to GenBank AccessionNo. NP_(—)003097 that functions in eye or neural development.

By “Sox2 nucleic acid sequence” is meant a polynucleotide encoding aSox2 polypeptide.

By “Stage Specific Embryonic Antigen-1 (SSEA-1) polypeptide is meant apolypeptide having at least 85% identity to GenBank Accession No.NP_(—)034372 or a human homolog thereof that acts as a marker forprimitive progenitor cells present in a mesodermal population.

By “SSEA-1 nucleic acid sequence” is meant a polynucleotide encoding aSSEA-1 polypeptide.

By “vascularization” is meant any biological process that increases theperfusion of tissue or organ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the results of flow cytometry analysis characterizingbone marrow mononuclear cells. The C57BL/6J BM-mononuclear cellfractions were analyzed by flow cytometry. At least 10,000 cells weregated in each case. FIG. 1A includes histograms showing thatCD31-expressing cells make up 30% of the cells, in contrast to otherwell-known stem cell markers which are expressed in very limited numbersof cells. FIG. 1B shows the results of FACS double stainingdemonstrating that CD34, c-kit, and flk-1 expressing cells are alsoexpressing CD31. FIG. 1C shows the results of FACS analysis afterlineage depletion. A positive portion of other stem cell markers alsoshare CD31 co-expression. CD31⁺ cells contain a high proportion ofhematopoetic (CD34+, c-kit+) and vasculogenic (Flk1+) stem cells.

FIG. 2 provides a graphical representation showing thesub-classification of bone marrow mononuclear cells divided by CD31.

FIG. 3 presents three histograms showing the results of FACS analysisfollowing CD31+/− selection with magnetic labeled beads. The purityafter magnetic sorting is confirmed by flow cytometry analysis againstCD31 (CD31-Neg: CD31-negatively selected cells; CD31-Pos:CD31-positively selected cells).

FIG. 4 provides the results of an RT-PCR showing embryonic geneexpression profiles of Oct-4 and Rex-4 in mouse embryonic stem (mES),mesenchymal stem cells (MSc), CD31⁺ and CD31⁻ cells. GAPDH expression isincluded as a control.

FIG. 5 is a graph showing the hematopoietic potential of CD31⁺cells.CD31 sorted cells from BM-MNCs were transplanted into lethallyirradiated mice. CD31⁺ cells rescued 100% of recipient mice and CD31⁻cells failed to provide a degree of protection from a lethal dose ofradiation (n=10, CD31+ and CD31− groups, respectively, n=5 for PBSgroup).

FIG. 6 shows microarray gene expression profiles for Akt-1, endothelialdifferentiation sphingolipid G-protein-coupled receptor 1 (Edg1), humangrowth factor (HGF), interleukin-18 (IL-18), neurophilin, nucleosidediphosphate linked moiety X-type motif 6 (Nudt6), platelet derivedgrowth factor-α, vascular endothelial growth factor-B (VEGF-B),chemokine (C—X—C motif) ligand 2 (cxc12), IL-1β, matrixmetalloprotease-9 (MMP-9), sphingosine kinase type 1 (sphk1),transforming growth factor-B3, tumor necrosis factor (TNF). Angiogenicmicroarray shows differential expressions on multiple genes between twogroups.

FIG. 7 shows fluorescent micrographs of an endothelial progenitor cell(EPC) culture assay. CD31⁺ cells gave rise to EPCs exclusively, comparedto CD31− cells. red: Ac-DiI-LDL, green: BS-1 lectin, blue: DAPI. n=7fields, each. *P<0.001, Whole vs. CD31-Neg; **P<0.001, Whole, CD31-Negvs. CD31-Pos

FIGS. 8A and 8B show the therapeutic efficacy of CD31⁺ cells. CD31⁺ celltransplantation showed greater efficacy to improve limb survival. FIG.8B is a graph showing the percent distribution of transplants havingfinal limb salvage among 3 groups. (n=7, each)

FIGS. 9A and 9B show limb perfusion at 14 days after celltransplantation. FIG. 9A includes 3 panels of color-coded images, whitehue indicates regions with maximum perfusion whereas blue representslowest perfusion area. FIG. 9B is a graph showing the quantification ofperfusion improvement. *P=0.629, **P=0.009, (n=3, each).

FIGS. 10A and B show the results of a histology analysis of ischemiclimb. Capillary densities were measured at 14 days. Administration ofCD31+ cells increased capillary density in ischemic tissue. FIG. 10Ashows representative lectin staining (left) and merged images with DAPI(right). FIG. 10B provides a quantitative analysis of capillary densityexpressed as the number of lection-positive cells per mm². *P=0.001, PBSvs. CD31-Pos; **P=0.002, CD31-Neg vs. CD31-Pos. (n=5, each)

FIGS. 11A-11C show the fate of CD31⁺ cells after 14 days transplantationinto ischemic limb. CD31⁺ cells from GFP expressing mice wereincorporated into vasculature as shown in FIGS. 11A-11C. Representativeimages stained for GFP (Anti-GFP antibody, green), lectin binding (red),and nuclear counterstain (DAPI, blue) demonstrate incorporation intoendothelial cells (arrow head) and pericytes location (arrow).

FIG. 12 shows angiogenesis-related gene expression levels in CD31⁺cells. Each dot represents relative expression levels of anangiogenesis-related gene in CD31⁺ cells relative to CD31− cells.

FIG. 13 shows the expression of lineage markers in CD31⁺ cells. Eachclosed bar represents the percentage of CD31+ cells among the specificmarker positive cells and each open bar represents the percentage ofspecific marker positive cells among CD31+ cells from murine bone marrowcells.

FIG. 14 shows limb perfusion after cell transplantation in a hind limbischemia model. One million cells were intramuscularly injected afterfemoral artery ligation. A. Limb perfusion at 14 days after celltransplantation. In these color-coded images, white hue indicatesregions with maximum perfusion whereas blue represents lowest perfusionareas. B. The quantification showed a perfusion improvement. *P=0.007(CD31+ vs PBS) (n=4, each)

FIG. 15 shows the histology analysis of an ischemic limb. Capillarydensities were measured at 14 days. Administration of CD31⁺ cellsincreased capillary densities in ischemic tissue. A. Representativelectin staining (red) and merged images with DAPI (blue). B.Quantitative analysis of capillary density was expressed as the numberof lection-positive cells per mm². *P=0.001, PBS vs. CD31+; **P=0.002,CD31− vs. CD31+. (n=5, each)

FIG. 16 shows the fate of CD31+ cells after transplantation intoischemic limb. CD31+ cells from GFP expressing mice incorporated intovasculature over 3-14 days. Representative images stained for GFP(Anti-GFP antibody, green), isolectin (red), and nuclear counterstaining(DAPI, blue) demonstrate incorporation into endothelial cell andpericyte locations.

FIG. 17 shows differentiation of CD31+ cells into endothelial cells invitro. One million CD31+ cells from murine bone marrow were plated on 2well chamber slides and cultured in EGM-2MV supplemented with 5% FBS and50 ng/ml VEGF (A). Cultured cells started to show a cobble stoneappearance at day 5 (B). Differentiation of CD31+ cells into ECs weredemonstrated by positive immunocytofluorescent staining for endothelialspecific markers.

FIG. 18 shows the characteristics of CD31+ and CD31− cells. Cells wereanalyzed with a FACStar flow cytometer. CD31+ cells were labeled withPE- or FITC-conjugated Abs against human CD11b, CD14, CD31, CD45, CD105,CD141, CD144, CD146 or isotype controls. Red lines, control Ig; greenline, specific Ab.

FIG. 19 shows the results of real time PCR analysis of specificendothelial and inflammatory genes in CD31⁺ and CD31⁻ cells.

FIG. 20 shows the characteristics of EPC and functional analysis ofperipheral blood CD31+ derived cells (A-B) Comparison of numbers ofadherent (AD) cells and colonies between CD31+ and CD31− cells, (C-D)EPC culture assay, (E) Colony-forming EPCs assay, (F) This panel showsthe counts of double-positive colonies grown from CD31+ and CD31− group,respectively (n=3; **P<0.01). (G) In vitro differentiation of CD31+cells into endothelial cells and (H) incorporation of CD31+ cells intothe HUVEC network.

FIG. 21 shows in vitro differentiation of CD31⁺ cells into vascular-liketubes after 4 weeks in EPC culture condition. (A) Sequential change ofdifferentiated CD31+ endothelial cells into vascular-like tubes. CD31+cells are capable of forming capillaries in vitro. (B-F)Immunohistochemistry of CD31+ cell-derived vascular-like tubes showsthat they aexpressed UEA-1 lectin and incorporated Dil-Ac-LDL like tubesgrown from endothelial cell lines. Nuclei counterstained with DAPI inblue.

FIG. 22 shows the results of real time PCR analysis of specificendothelial and inflammatory genes in endothelial cell differentiatedCD31+ cells. CD31+ cells were cultured in EPC culture medium for 4weeks. Non-adherent cells were discarded and only adherent cells wereanalyzed.

FIG. 23 shows the results of in vivo vasculogenesis of CD31 cells in anischemic limb of a nude mouse.

FIG. 24 shows the results of real time PCR analysis of multipleangiogenic factors following CD31⁺ transplantation into the ischemichindlimb of athymic nude mice.

FIG. 25 shows transdifferentiation of CD31+ cells into endothelialcells. (A and B are images from replicate samples) Immunofluorescencestaining images shows that human peripheral blood CD31 cells aretransdifferentiated into endothelial cells lineage. Tissue sections arefrom hind limb harvested at 2 weeks from animals with hindlimb ischemiafollowed by the intramuscular injection of Dil-labeled CD31 cells (red).Sections were stained for ILB-4 (Green) which is endothelial cellmarker. For nuclei detection, DAPI (blue) was counterstained. Bar: 50 umThe “merged” panel shows cells double positive for DiI and ILB-4 binding(green), appearing yellow on merged images.

FIG. 26 shows the results of quantitative analysis of TUNEL-positiveCD31⁺ and CD31− cells. (A-B)

FIG. 27 shows the results of realt time PCR analysis of human bonemarrow CD31⁺ cells.

FIG. 28 shows the results of flow cytometry analyses for CD31 expressionon BM cells, Lin⁻cells and Lin⁻c-kit+Sca-1+ cells. A: FACS analysis forCD31 expression on mouse BM cells after red blood cell (RBC) lysis.Black line is isotype control. Green line is specific mAb. B: CD31expression on Lin⁻ cells. Black line is isotype control. Green line isspecific mAbs. D: CD31 expression on Lin⁻ c-kit+Sca-1+ cells. Red lineis isotope control and green line is specific mAb.

FIG. 29 shows FACS analysis for expression of stem cell markers,including Sca-1, Flk-1 and c-kit. BM cells were partiallylineage-depleted by staining of BM cells with mouse biotinalyted lineagemAbs followed by anti-biotin microbeads. Partially depleted Lin⁻ cellswere incubated with mouse mAbs, including APC-lineage cocktail,PE-Sca-1, PE-c-kit, PE-Flk-1, FITC-CD31 and CD45, and then analyzed byflow cytometry.

FIG. 30 shows the results of a clonogenic assay of CD31 expressingcells. A: clonogenic assay of CD31⁺ cells. **, p<0.001, CD31⁺ vs.BMMNCs, n=6 for each group. ND, not detectable. B: Clonogenic assay ofLin⁻CD31⁺ cells. **, p<0.001, Lin⁻CD31⁺ vs. Lin⁻CD31−, n=6 for eachgroup.)

FIG. 31 shows the results of in vivo culture of CD31 expressing cells.FIG. 4A: culture of CD31+ cells with TPO, FLT3L and SCF. B: culture ofLin−CD31+ cells with SCF, TPO, FLT3L. C: flow cytometry analyses ofcultured Lin−CD31+ cells for expression of stem cell markers, indicatingthat the Lin−CD31+ cells, as a hematopoietic stem and progenitor cellscan be expanded efficiently in vitro. D: Multiple-lineagedifferentiation of Lin−CD31+ cells. E: MoFlo sorting for Lin−CD31+Sca-1+ cells. F: In vitro culture of Lin−CD31+ Sca-1+ cells. G. multiplelineage differentiation of Lin−CD31−Sca-1+ cells.

FIG. 32 shows the results of FACS analysis for in vivo BM repopulation.A: reconstitution of total BM transplantation chimeras 5 weeks after BMtransplantation. (*, P<0.05, lin−CD31+ vs. Lin−CD31− cells, n=5 pergroup). B: a representative dot blot analysis of C57BL/6J micetransplanted with either Lin−CD31+ or Lin−CD31− showing reconstitutionsof total BM transplantation chimeras.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods related to theisolation, culture and therapeutic use of CD31-expressing cells. Theinvention is based, at least in part, on the discovery that CD31 orPECAM-1, which has been generally regarded as an endothelial cellmarker, is a surface epitope common to most stem or progenitor cellsthat have been shown to have therapeutic effects in cardiovascularregeneration or repair. Moreover, lineage negative, CD31 positive cellsinclude pluripotent hematopoietic stem cells which can differentiateinto a variety of lineages of hematopoietic cells and are useful fortreating various hematologic diseases. Furthermore, developmentalstudies using embryonic stem cells have demonstrated that CD31 is notmerely a marker for endothelial lineage differentiation but a maker formultipotent stem cells.

Accordingly, BM-derived CD31-positive cells were characterized and thetherapeutic use of these cells has been determined. BM-derivedCD31-positive cells are useful for regenerating or repairing tissuesaffected by a variety of diseases characterized by an increase in celldeath or a decrease in cell number or function. In particular, CD31⁺cell transplantation is useful for repairing ischemic limb and heart anduseful for reconstituting hematopoietic cells following whole bodyirradiation required in bone marrow transplantation. Such cells may alsobe used for the treatment or prevention of cardiovascular diseases, suchas myocardial infarction, congestive heart failure, peripheral vascularobstructive diseases, various types of peripheral neuropathies includingbut not limited to diabetic, ischemic, toxic or chemical-inducedneuropathies, stroke (cerebrovascular diseases), liver failure, renalfailure, islet cell transplantation, bone and joint diseases or anydegenerative disease which requires stem cell therapy.

CD31

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kDa typeI transmembrane glycoprotein (GP) that was originally described as theendothelial cell equivalent of platelet membrane GPIIa (the integrin β1subunit) and a myeloid differentiation antigen. The common identity ofthese previously disparate entities as PECAM-1 or CD31 was finallyestablished in 1990 upon its cloning by 3 different groups⁴⁰⁻⁴². TheCD31 antigen has been known to present on the cell-cell junctions ofendothelial cells and surface of hematopoietic cells includingmonocytes, granulocytes, natural killer cells, naïve T and cells andplatelets⁴³⁻⁴⁶. In general, CD31 has served as an immunochemical markerto identify endothelial cells in histological sections or to markangiogenic blood vessels.

Recently other roles for CD31 have been suggested. CD31 may work as akey mediator of the migration of cells in the process of angiogenesis.For example, an antibody against murine CD31 inhibits tumor angiogenesisin a mouse model as well as capillary growth into subcutaneouslyimplanted gels supplemented with bFGF⁴⁷. The preliminarycharacterization of CD31-deficient mice (CD31-KO) also focused on afunctional evaluation of CD31 in the context of inflammatoryadhesion-dependent cascades and not in the context of anangio-vasculogensis⁴⁸. Interestingly, CD31-KO mice are viable, remainhealthy, and exhibit no obvious vascular developmental defects duringembryonic development. In vascular biology, CD31 mediates the arteriolardilation in response to wall shear stress in an nitrous oxide(NO)-dependent manner⁴⁹. Previous studies failed to recognize that CD31could serve as a stem cell marker or function in postnatal adultvasculogenesis/myogenesis. As reported in more detail below, CD31 servesas a comprehensive epitope that is expressed in various subsets ofhemangioblastic cells in adult bone marrow (BM).

The amino acid sequence of an exemplary CD31 polypeptide is provided atGenBank Accession No. NP_(—)000433.

(SEQ ID NO.: 1) 1 mqprwaqgat mwlgvlltll lcsslegqen sftinsvdmk slpdwtvqngknltlqcfad 61 vsttshvkpq hqmlfykddv lfynissmks tesyfipevr iydsgtykctvivnnkektt 121 aeyqllvegv psprvtldkk eaiqggivrv ncsvpeekap ihftieklelnekmvklkre 181 knsrdqnfvi lefpveeqdr vlsfrcqari isgihmqtse stkselvtvtesfstpkfhi 241 sptgmimega qlhikctiqv thlaqefpei iiqkdkaiva hnrhgnkavysvmamvehsg 301 nytckvessr iskvssivvn itelfskpel essfthldqg erlnlscsipgappanftiq 361 kedtivsqtq dftkiasksd sgtyictagi dkvvkksntv qivvcemlsqprisydaqfe 421 vikgqtievr cesisgtlpi syqllktskv lenstknsnd pavfkdnptedveyqcvadn 481 chshakmlse vlrvkviapv devqisilss kvvesgediv lqcavnegsgpitykfyrek 541 egkpfyqmts natqafwtkq kaskeqegey yctafnranh assvprskiltvrvilapwk 601 kgliavviig viialliiaa kcyflrkaka kqmpvemsrp avpllnsnnekmsdpnmean 661 shyghnddvr nhamkpindn keplnsdvqy tevqvssaes hkdlgkkdtetvysevrkav 721 pdavesrysr tegsldgt

Lineage negative CD31 is a marker for a population of multipotent stemcells. CD31⁺ cells were isolated with the use of magnetic cell isolationsystem and antibodies that recognized CD31 and a cocktail of varioushematopoietic lineage markers. In addition, methods for maintainingCD31⁺ cells in culture dishes. Compared to the other cells, lineagenegative CD31 positive cells were easier to isolate and have similar orsuperior multipotency and therapeutic capacity. Lineage negative,CD31-positive bone marrow cells are multipotent and possess angiogenicand regenerative capacity. These cells can be therapeutically employedin a variety of cardiovascular and other diseases.

Ischemic Disease

Ischemia refers to a condition characterized by oxygen deficiency.Typically ischemia is related to insufficient circulation. Ischemia canaffect virtually any organ in the body, including but not limited tomuscle tissues, organs, and neural tissues. Neural ischemias includetransient ischemia, stroke, and reperfusion injury. In intestinalischemia blood supply to the intestine is reduced resulting in tissuedamage. Ischemic colitis involves an area of inflammation (irritationand swelling) caused by interference with the blood flow to the colon.Cardiac ischemia is associated with myocardial infarction and congestiveheart failure. Limb ischemia is associated with peripheral vascularobstructive disease.

Cardiovascular Function

Cardiac conditions, such as myocardial infarction, congestive heartfailure, peripheral vascular obstructive disease, cardiac hypertrophy,reduced systolic function, reduced diastolic function, maladaptivehypertrophy, heart failure with preserved systolic function, diastolicheart failure, hypertensive heart disease, aortic and mitral valvedisease, and pulmonary valve disease are associated withischemia-related tissue damage and/or reduced cardiac function.Compositions of the invention containing a CD31⁺ cell may be used toenhance angiogenesis in a cardiac tissue or to increase cardiac functionin a subject having reduced cardiac function. Desirably, cardiacfunction is increased by at least 5%, 10% or 20%, or even by as much as25%, 50% or 75%. Most advantageously, cardiac function is increasedrelative to the function of an untreated control. Treatments thatincrease cardiac function are useful in the methods of the invention.

Any number of standard methods are available for assaying cardiovascularfunction. Preferably, cardiovascular function in a subject (e.g., ahuman) is assessed using non-invasive means, such as measuring netcardiac ejection (ejection fraction, fractional shortening, andventricular end-systolic volume) by an imaging method suchechocardiography, nuclear or radiocontrast ventriculography, or magneticresonance imaging, and systolic tissue velocity as measured by tissueDoppler imaging. Systolic contractility can also be measurednon-invasively using blood pressure measurements combined withassessment of heart outflow (to assess power), or with volumes (toassess peak muscle stiffening). Measures of cardiovascular diastolicfunction include ventricular compliance, which is typically measured bythe simultaneous measurement of pressure and volume, early diastolicleft ventricular filling rate and relaxation rate (can be assessed fromecho-Doppler measurements). Other measures of cardiac function includemyocardial contractility, resting stroke volume, resting heart rate,resting cardiac index (cardiac output per unit of time [L/minute],measured while seated and divided by body surface area [m²])) totalaerobic capacity, cardiovascular performance during exercise, peakexercise capacity, peak oxygen (O₂) consumption, or by any other methodknown in the art or described herein. Measures of vascular functioninclude determination of total ventricular afterload, which depends on anumber of factors, including peripheral vascular resistance, aorticimpedance, arterial compliance, wave reflections, and aortic pulse wavevelocity,

Methods for assaying cardiovascular function include any one or more ofthe following: Doppler echocardiography, 2-dimensional echo-Dopplerimaging, pulse-wave Doppler, continuous wave Doppler, oscillometric armcuff, tissue Doppler imaging, cardiac catheterization, magneticresonance imaging, positron emission tomography, chest X-ray, X-raycontrast ventriculography, nuclear imaging ventriculography, computedtomography imaging, rapid spiral computerized tomographic imaging, 3-Dechocardiography, invasive cardiac pressures, invasive cardiac flows,invasive cardiac pressure-volume loops (conductance catheter),non-invasive cardiac pressure-volume loops.

Neuropathy

Neuropathies are pathologies that disrupt neural function. There is amarked reduction of the microvasculature (vasa nervorum) in diabeticperipheral neuropathy, particularly. Impaired angiogenesis, inparticular, attenuation of the vasa nervorum, has been noted in modelsof diabetes. Microvascular insufficiency and neurotrophic factordeficiency plays a role in the development and progression of diabeticperipheral neuropathy, as does ischemia in diabetic nerves (Dyck P J,1989, Neurology; Steven E J, 1994, Diabetologia), inactivation ofproteins critical to neural function (Cullum N A, 1991, Diabetologia)and altered neural polyol metabolism (Greene D A, 1987, NEJM; Cameron NE, 1994, Diabetes Metab. Rev). Diabetic neuropathy can be reversed byagents promoting angiogenesis such as VEGF-1 and ±2 (Schratzberger etal., J Clin Invest. May 2001; 107(9):1083-1092), sonic hedgehog (SHh)(Kusano et al., Arterioscler Thromb Vasc Biol. November 2004;24(11):2102-2107), and a statin (Ii et al., Circulation. Jul. 5, 20052005; 112(1):93-102). Furthermore, promising results from a pilotclinical trial (Simovic et al., Arch Neurol. May 2001; 58(5):761-768;Isner et al., Hum Gene Ther. Aug. 10, 2001; 12(12):1593-1594) using aplasmid encoding human VEGF (phVEGF) via gene therapy approach forpatients with DPN also support the importance of vascular supply in thepathogenesis of DPN.

Exemplary neuropathies include but are not limited to diabeticneuropathy, ischemic neuropathy, toxic neuropathy, diabetic dementia.Symptoms of neuropathy vary depending on whether the affected nerves aresensory, motor, or autonomic. Neuropathy can affect any one or acombination of all three types of nerves. Typically, symptoms ofneuropathy include pain, loss of sensation, or inability to controlmuscles. Methods of assaying neuropathy include electromyography, nerveconduction velocity tests, nerve biopsy, and other standard clinicalassays for neurological function. Peripheral neuropathy is characterizedby an abnormal neurological exam, subjective symptoms, abnormalbiothesiometry, or abnormal nerve conduction study. Autonomic neuropathyis characterized by an abnormal R—R interval, orthostatic hypotension,or resting tachycardia.

Neuropathy may be caused by a hereditary disorder (e.g.,Charcot-Marie-Tooth disease, Friedreich's ataxia), an infectious orinflammatory conditions (e.g., rheumatoid arthritis, lyme disease,AIDs), exposure to agents that are toxic to neurons (e.g., heavy metals,such as lead), or systemic or metabolic disorders, such as diabetes.

As reported in more detail below, the present invention providescompositions and methods for treating or preventing neuropathies,including diabetic neuropathy, by increasing angiogenesis in themicrovasculature of nerves. The invention is based, in part, on thediscovery that the local implantation of multipotent stem cells, such asendothelial progenitor cells, mesenchymal stem cells, and peripheralblood mononuclear cells, increases vascularity, conduction velocity,cytokine or therapeutic polypeptide expression, Schwann and endothelialcell proliferation, and decreases apoptosis in nerves affected byneuropathy.

Therapeutic and Prophylactic Methods

The invention provides for the treatment of diseases and disordersassociated with an increase in cell death or a decrease in cell number.In particular, CD31⁺ expressing cells are useful for the treatment ofliver failure, renal failure, islet cell transplantation, bone and jointdiseases or any degenerative disease which could benefit from stem celltherapy.

Many diseases associated with a deficiency in cell number arecharacterized by an increase in cell death. For example, the inventionprovides compositions for the treatment of diabetic patients who lacksufficient levels of insulin due to a decrease in the number or activityof insulin producing pancreatic cells. Such diseases include, but arenot limited to, neurodegenerative disorders, stroke, myocardialinfarction, or ischemic injury. Injuries associated with trauma can alsoresult in a deficiency in cell number in the area sustaining the wound.Methods of the invention ameliorate such diseases, disorders, orinjuries by generating cells that can supplement the deficiency. Suchcells are generated from the transdifferentiation of a CD31⁺ bone marrowcell to a cell type of interest (e.g., the transdifferentiation of aCD31⁺ expressing cell to an endothelial cell, an insulin producing cell,a bone cell, a liver cell, a renal cell) or by promoting theregeneration of a cell, tissue, or organ.

In one embodiment, a CD31⁺ expressing stem cell of the invention isadministered to a cell, tissue, or organ in situ to ameliorate adeficiency in cell number. Alternatively, the CD31⁺ expressing stem cellis administered to a tissue in vitro and then the CD31⁺ expressing stemcell are administered to the patient to ameliorate a disease, disorder,or injury. In one embodiment, a CD31⁺ expressing stem cell is deliveredlocally to a site where an increase in angiogenesis or tissue repair isdesired. Administration may be by any means sufficient to result in atherapeutic effect. In various embodiments, CD31⁺ expressing stem cellare administered by local injection to a site of disease or injury, bysustained infusion, or by micro-injection under surgical conditions(Wolff et al., Science 247:1465, 1990). In other embodiments, the CD31⁺expressing stem cells are administered systemically to a tissue or organof a patient having a deficiency in cell number that can be amelioratedby cell regeneration.

CD31⁺ cells having the potential to differentiate into endothelial cells(e.g., multipotent stem cells, endothelial progenitor cells, mesenchymalstem cells, mononuclear cells, or progenitors or progeny thereof) can beused in a variety of therapeutic or prophylactic applications.Accordingly, methods of the invention relate to, among other things, theuse of multipotent stem cells, endothelial progenitor cells, mesenchymalstem cells, mononuclear cells, or progenitors or progeny thereof for thetreatment or prevention of ischemic diseases (e.g., myocardialinfarction, limb ischemia, stroke, transient ischemia, reperfusioninjury) neuropathies (e.g., diabetic peripheral neuropathy, toxicneuropathy), liver failure, renal failure, diabetes, bone and jointdiseases or any degenerative disease which requires stem cell therapy.

In one embodiment, the present invention provides methods for treatingneuropathy comprising providing a cell having the potential todifferentiate into an endothelial cell (e.g., multipotent stem cells,endothelial progenitor cells, mesenchymal stem cells, mononuclear cells,or progenitors or progeny thereof) to a subject in need thereof, whereinthe cell engrafts into a tissue (e.g., a neural tissue) and enhancesangiogenesis in a neural tissue of interest. In one embodiment, thepresent invention provides methods for treating neuropathy comprisingproviding a cell having the potential to differentiate into anendothelial cell (e.g., multipotent stem cells, endothelial progenitorcells, mesenchymal stem cells, mononuclear cells, or progenitors orprogeny thereof) to a subject in need thereof, wherein the cell engraftsinto the microvasculature of a neural tissue of interest and increasesangiogenesis, vascularity, or the biological function of the neuraltissue. In yet another embodiment, the method provides a cell having thepotential to differentiate into an endothelial cell (e.g., multipotentstem cells, endothelial progenitor cells, mesenchymal stem cells,mononuclear cells, or progenitors or progeny thereof) to a subject inneed thereof, wherein the cell augments a humoral response in the neuraltissue sufficient to exert a therapeutic effect (e.g., an increase inparacrine factors, neurotrophic factors, angiogenic factors, or anincrease in angiogenesis).

The present invention also provides methods for restoring neuralfunction in a diabetic subject having a loss of neural function (e.g.,motor or sensory deficit), comprising providing a cell having thepotential to differentiate into an endothelial cell and a neural cell(e.g., multipotent stem cells, endothelial progenitor cells, mesenchymalstem cells, mononuclear cells, or progenitors or progeny thereof) to thesubject to enhance neural function.

In another embodiment, the invention provides methods for treating liverfailure, renal failure, or diabetes comprising providing a cell havingthe potential to transdifferentiate into a liver cell, renal cell, orinsulin producing cell (e.g., a pancreatic islet cell) to a subject inneed thereof, wherein the cell engrafts into a tissue of the subject andrepairs or regenerates the tissue.

In general, the sample comprising the CD31⁺ cells can be pretreated in awide variety of ways. Generally, once collected, the cells can beadditionally concentrated, if this was not done simultaneously withcollection or to further purify and/or concentrate the cells. The cellsmay be washed, counted, and resuspended in buffer transferred to asterile, closed system for further purification and activation.

The CD31⁺ cells are generally concentrated for treatment, using standardtechniques in the art. In a preferred embodiment, the leukophoresiscollection step results in a concentrated sample of CD31⁺ cells, in asterile leukopak, that may contain reagents or doses of a suppressivecomposition. Generally, an additional concentration/purification step isdone, such as Ficoll-Hypaque density gradient centrifugation as is knownin the art. Separation or concentration procedures include but are notlimited to magnetic separation, using antibody-coated magnetic beads,affinity chromatography, cytotoxic agents, either joined to a monoclonalantibody or used with complement, “panning”, which uses a monoclonalantibody a to a solid matrix. Antibodies attached to solid matrices,such as magnetic beads, agarose beads, polystyrene beads, follow fibermembranes and plastic surfaces, allow for direct separation. Cells boundby, antibody can be removed or concentration by physically separatingthe solid support from the cell suspension. The exact conditions andprocedure depend on factors specific to the system employed. Theselection of appropriate conditions is well within the skill in the art.

Antibodies may be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with a fluorescence activated cell sorter (FACS), to enable cellseparation. Any technique may be employed as long as it is notdetrimental to the viability of the desired cells.

In a preferred embodiment, the CD31⁺ cells are separated in anautomated, closed system such as the Nexell Isolex 300i Magnetic CellSelection System. Generally, this is done to maintain sterility and toinsure standardization of the methodology used for cell separation,activation and development of suppressor cell function.

Once purified or concentrated the cells may be aliquoted and frozen,preferably, in liquid nitrogen or used immediately as described below.Frozen cells may be thawed and used as needed. Cryoprotective agents,which can be used, include but are not limited to dimethyl sulfoxide(DMSO) (Lovelock, J. E. and Bishop, M. W. H., 1959, Nature183:1394-1395; Ashwood-Smith, M. J., 1961, Nature 190:1204-1205),hetastarch, glycerol, polyvinylpyrrolidine (Rinfret, A. P., 1960, Ann.N.Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter, H. A. andRavdin, R. G., 1962, Nature 196:548), albumin, dextran, sucrose,ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe, A. W., etal., 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose,choline chloride (Bender, M. A., et al., 1960, J. Appl. Physiol.15:520), amino acids (Phan The Tran and Bender, M. A, 1960, Exp. CellRes. 20:851), methanol, acetamide, glycerol monoacetate (Lovelock. J.E., 1954, Biochem. J. 56:265), and inorganic salts (Phan The Tran andBender, M. A., 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tranand Bender, M. A., 1961, in Radiobiology Proceedings of the ThirdAustralian Conference on Radiobiology, Ilbery, P. L. T., ed.,Butterworth, London, p. 59). Typically, the cells may be stored in 10%DMSO, 50% serum, and 40% RPMI 1640 medium. Methods of cell separationand purification are found in U.S. Pat. No. 5,888,499, which isexpressly incorporated by reference.

In a preferred embodiment, the CD31⁺ cells are then washed to removeserum proteins and soluble blood components, such as autoantibodies,inhibitors, etc., using techniques well known in the art. Generally,this involves addition of physiological media or buffer, followed bycentrifugation. This may be repeated as necessary. They can beresuspended in physiological media, preferably AIM-V serum free medium(Life Technologies) (since serum contains significant amounts ofinhibitors of TGF-β) although buffers such as Hanks balanced saltsolution (HBBS) or physiological buffered saline (PBS) can also be used.

Generally, the cells are then counted; in general from 1×10⁹ to 2×10⁹white blood cells are collected from a 5-7 liter leukophoresis step.These cells are brought up to roughly 200 mls of buffer or media.

Compositions comprising peripheral blood derived stem cells or theirprogenitors can be provided directly to a tissue of interest.Alternatively, compositions comprising stem cells or their progenitorscan be provided indirectly to the tissue of interest, for example, byadministration into the circulatory system or injection into a skeletalor cardiac muscle.

CD31⁺ Cells Engraftment

As described in more detail below, the present invention providesmethods for preventing or treating ischemic diseases (e.g., myocardialinfarction, limb ischemia, stroke, transient ischemia, reperfusioninjury) neuropathies (e.g., diabetic peripheral neuropathy, toxicneuropathy), liver failure, renal failure, diabetes, bone and jointdiseases or any degenerative disease which requires stem cell therapy bylocally or systemically administering a cell having the potential todifferentiate into an endothelial cell, endothelial progenitor cell,mesenchymal stem cell, mononuclear cell, liver cell, renal cell, neuralcell, skin cell, cells derived from eye tissue, insulin producing cell,or progenitors or progeny thereof directly or indirectly to the tissuein need of therapy. In one embodiment, this administration results inthe incorporation of the cell into a tissue (e.g., a tissue comprisingdifferentiated somatic cells, extracellular matrix, and supportingcells, such as cells of the vasculature that supply the tissue). Withoutwishing to be bound by theory, it is likely that cells of the inventionhome to tissue that lacks sufficient vascularity and are incorporatedinto that tissue. In one embodiment, the cells differentiate into matureendothelial cells and contribute to the vasculature of the neural,skeletal muscle, cardiac muscle or skin tissue. Methods for detectingdifferentiated endothelial cells are known in the art, see, for example,U.S. Pat. No. 5,980,887 and WO 99/45775, which describe methods fordetecting and monitoring endothelial cell function. A preferred assayinvolves detection of EC specific markers (e.g., VE-Cadherin, CD34,Flk-1, Tie2 and CD31, VonWillebrand Factor or factor 8).

CD31⁺ Cells

CD31⁺ stem cells are isolated by standard means known in the art for theseparation of stem cells from the bone marrow, peripheral blood andumbilical cord blood. In particular, methods of the invention providefor the isolation of a CD31⁺ expressing stem cell.

The invention encompasses CD31+ cells that are uncultured or cultured.“Uncultured” as it refers to CD31+ cells means a population of CD31+cells that has not been cultured as defined herein. “Uncultured” CD31+cells means the total population of mononuclear cells derived from anyone of bone marrow, peripheral blood or umbilical cord blood.“Uncultured” CD31+ cells also means cells that have been subjected to astep wherein total CD31+ cells are isolated, for example, magnetic cellsorting.

As used herein, a “cultured CD31⁺ cell” is a cell that has been grown invitro. For example, subjected to a step where total CD31″ cells areisolated and at least one additional culture step is carried out, forexample, expansion, wherein the number of cells is increased while thesurface marker expression is unchanged, or undergoes a low level ofchange; or differentiation, wherein cells are cultured under conditionswhich promote formation of any one of hematopoietic stem cells(non-adherent), endothelial progenitor cells (adherent and non-adherent)and mesenchymal progenitor cells (adherent) is performed.

In one embodiment, “culturing” includes a step of selecting for lin⁻cells.

In one embodiment, CD31⁺ expressing stem cells useful in the methods ofthe invention are obtained from the bone marrow of a human patient.Typically, the method includes at least one or more of the followingsteps:

-   -   a) collecting bone marrow cells from a mammal (e.g., a young        adult), where the cells have a size of less than about 100        microns, less than about 50 microns, or about 40 microns or        less,    -   b) culturing (expanding) the collected cells in medium under        conditions that select for adherent cells,    -   c) selecting the adherent cells and expanding those cells in        medium to semi-confluency,    -   d) serially diluting the cultured cells into chambers with        conditioned medium, the dilution being sufficient to produce a        density of less than about 1 cell per chamber to make clonal        isolates of the expanded cells; and    -   e) culturing (expanding) each of the clonal isolates and        selecting chambers having expanded cells to make the population        of isolated bone marrow cells.

In another embodiment, hemangioblastic cells having the potential todifferentiate into hematopoietic cells or endothelial cells (e.g.,multipotent stem cells, endothelial progenitor cells, mononuclear cells,mesenchymal stem cells, and their progenitor or progeny cells) areobtained by extracting fresh unprocessed bone marrow cells from youngdonors. The cells are typically separated from blood cells bycentrifugation, hemolysis and related standard procedures describedherein. The bone marrow cells are washed in an acceptable buffer such asPBS and filtered to collect cells having a size less that about 100microns, less than about 50 microns, or about 40 microns. Methods forsize selection are known in the art. In one embodiment, a standard nylonfilter is used. Once isolated, cells of the selected size are grown on acomplete culture medium with low or high glucose (e.g., DMEM) thatcontains a rich source of growth factors and cytokines. Fetal bovineserum (FBS) is typically used in the culture medium. Cells are cultured(i.e. expanded) for less than about two weeks, preferably about a weekor less such as four to six days. The conditioned medium is thenreplaced with fresh medium; adherent cells are removed from the culturedishes and resuspended in fresh medium to select cells for expansion.The selected cells are grown to semiconfluency (between 50% to 90%confluent) and again, adherent cells are selected. Such cells are thenreseeded in complete medium in a tissue culture flask at a density ofabout 10⁴ cells per centimeter. After the cells reach semiconfluency,they are reseeded (serially) into the flasks at the same or similardensity. The cultures are preferably passaged more than one time,typically less than five times and preferably about two times tocontinue selection for expanding cells. Selected cells are then seriallydiluted into single well chambers (e.g., standard 96 well plate) at adensity of less than about 1 cell per chamber, preferably ½ a cell perchamber. Preferably, the cells are cultured with conditioned media topromote growth to sub confluence (i.e. less then 50% confluent). Wellswith expanded cell clones are expanded and replated as needed.

If desired, cell clones are selected that express a normal or an alteredlevel (e.g., increased or decreased level) of at least one of thefollowing markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4,GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor, MHCclass II receptor or other cell marker as described herein as comparedto CD31− cells. Methods for performing the selection include any of thesuitable assays disclosed herein. In embodiments in which larger amountsof cells are needed a more automated or semi-automated method will oftenbe preferred such as fluorescence activated cell sorting (FACS).Selected cells desirably are able to be propagated in culture for longperiods of time without becoming polyploidy or losing multipotency.

Peripheral blood derived cells of the invention, their progenitors ortheir progeny, are obtained by methods known in the art, includingmethods for harvesting umbilical cord blood. In general, peripheralblood mononuclear cells (PBMCs) are taken from a patient using standardtechniques. By “peripheral blood mononuclear cells” or “PBMCs” herein ismeant lymphocytes (including T-cells, B-cells, NK cells), monocytes andstem cells. Prior to harvest, patients may be treated with agents knownin the art to increase mobilization of stem cells from the bone marrowinto the peripheral blood. Mobilizing agents include but are not limitedto GCSF or GMCSF. In some embodiments of the invention, only PBMCs aretaken, either leaving or returning red blood cells and polymorphonuclearleukocytes to the patient. This is done as is known in the art, forexample using leukophoresis techniques. In general, a 5 to 7 literleukophoresis step is done, which essentially removes PBMCs from apatient, returning the remaining blood components. Collection of thecell sample is preferably done in the presence of an anticoagulant suchas heparin, as is known in the art.

Peripheral blood derived stem cells of the invention can, if needed, bepurified from peripheral blood, including umbilical cord blood. Humanumbilical cord blood (“cord blood”) is a rich source of hematopoieticstem cells, hemangioblasts, endothelial progenitor cells, mesenchymalstem cells (MSCs). CD31+ cells are first isolated from peripheral bloodor cord blood mononuclear cells by density gradient method andimmuno-sorting such as MACS or FACS. Methods of isolating such cells areknown in the art. Briefly, a 1 ml portion of umbilical cord is placed ina well containing RPMI and 20% FBS. The matrix cells migrate out fromthe cord and adhere to the plastic well. Such cells have a fibroblastmorphology. The supernatant and tissue are discarded after several daysin culture. The cells remaining in the well are trypsinized andtransferred to a secondary culture for expansion. See, for example,Connealey et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 9836-9841,September 1997; and Meagher and Klingemann et al., J Hematother StemCell Res. 2002 June; 11(3):445-8. J Hematother Stem Cell Res. 2002 June;11(3):445-8. While particular examples are directed to bonemarrow-derived cells, one skilled in the art appreciates that anyhematopoietic stem cell may be used in the methods of the invention.

Therefore, cells that can be used in the methods of the invention cancomprise a purified sub-population of cells including, but not limitedto stem cells, or any cell having the ability to give rise toendothelial cells under suitable conditions in vitro or in vivo.“Suitable conditions” are empirically determined by culturing orimplanting a cell of the invention then subsequently identifyingendothelial cells in the culture or implant (e.g., cells havingendothelial morphology, function, or expressing one or more endothelialcell markers). Purified cells can be collected and separated, forexample, by flow cytometry. Peripheral blood derived cells of theinvention can be autologous (obtained from the subject) or heterologous(e.g., obtained from a donor). Heterologous cells can be providedtogether with immunosuppressive therapies known in the art to preventimmune rejection of the cells.

Purified peripheral blood derived cells or their progenitors can beobtained by standard methods known in the art, including cell sorting byMACS or FACs. Isolated peripheral blood can be sorted using flowcytometers known in the art (e.g., a BD Biosciences FACScaliburcytometer) based on cell surface expression of Sca-1 (van de Rijn etal., (1989) Proc. Natl. Acad. Sci. USA 86, 4634-4638) and/or c-Kit(Okada et al., (1991) Blood 78, 1706-1712); (Okada et al., (1992) Blood80, 3044-3050) following an initial immunomagnetic bead column-basedfractionation step to obtain lineage-depleted (lin⁻) cells (Spangrude etal., (1988) Science 241, 58-62); (Spangrude and Scollay, (1990) Exp.Hematol. 18, 920-926), as described (Shen et al., (2001) J. Immunol.166, 5027-5033); (Calvi et al., (2003) Nature 425, 841-846).

For serial passage-based enrichment of peripheral blood stem cells ortheir progenitors in-vitro (Meirelles and Nardi, (2003) Br. J. Haematol.123, 702-711); (Tropel et al., (2004) Exp. Cell Res. 295, 395-406),isolated peripheral blood can be plated on plastic in Dulbecco'smodified Eagle's medium (Fisher Scientific, Pittsburgh, Pa.) with 10%fetal bovine serum (Hyclone, Logan, Utah), penicillin, streptomycin,L-glutamine and amphotericin-B. About forty-eight hours after theinitial plating, the supernatants containing non-adherent cells can beremoved and replaced with fresh culture medium after gentle washing. Thecultures can then be maintained and passed once confluence is reached(e.g., for a total of about three times over the span of about 6 weeks)at which time the cultures can be terminated to collect adherent cellsfor analysis.

In one embodiment, a method for isolating stem cells of the invention(e.g., CD31⁺ expressing cells) includes generation of a fraction thatcomprises cells expressing or having an altered level of expression(i.e., increased or decreased) of any one or more of the followingmarkers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5,Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class IIreceptor as determined by standard cell marker detection assay ascompared to CD31⁻ cells. Additional selection means based on the uniqueprofile of gene expression can be employed to further purify populationsof cells capable of generating an endothelial cell. Compositionscomprising an endothelial progenitor cell can be isolated andsubsequently purified to an extent where they become substantially freeof the biological sample from which they were obtained.

CD31⁺ multipotent stem cells and their progenitor cells or progeny canbe obtained from bone marrow or peripheral blood and then expanded inculture. Thus, the progenitor cells can be cells having an “expansionphenotype” characterized by expressing or having an altered level ofexpression (i.e., increased or decreased) of any one or more of thefollowing markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4,GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHCclass II receptor as compared to CD31⁻ cells. Alternatively, adifferentiated endothelial cell expresses one or more characteristicendothelial cell markers that provide for its identification, suchmarkers include, but are not limited to, VE-Cadherin, CD34, Flk-1, Tie2and CD31, VonWillebrand Factor or factor 8.

Fractionation of CD31⁺ Stem Cells

The invention provides for the isolation, culture, and expansion ofCD31⁺ cells. As reported herein, CD31 acts as a marker for pluripotentstem cells. There exist several methods to fractionate desired BM stemor progenitor cells for the purpose of tissue regeneration. Flowcytometry or magnetic-labeled cell sorting is one of the most popularmethods. It is based on the expression of a combination of antigenmarkers. Markers used in flow cytometry include CD31⁺, as describedherein. Other markers known in the art include, but are not limited to,c-kit, Sca-1, CD34, Flk-1. CD34⁺ cells or c-kit⁺ cells exist in lownumbers in BM (less than 1%), so the mobilization process, which maycause serious adverse effects (restenosis after vascular interventionand atherosclerotic plaque rupture because of the inflammatoryresponse), is required to obtain a sufficient number of cells to be usedfor cell therapy.

Alternative approaches to FACS depend on one or more physiologicproperties of stem cells; the side population isolation via preferentialefflux of the DNA binding dye Hoechst 33342, which was originallyinvented for long term repopulating hematopoietic stem cell isolation inBM²³, based on ABC/G2 transporters, ATP binding cassette transportersexpressed selectively on the surface of primitive stem or progenitorcells in a variety of sources including liver²⁵, skeletal muscle²⁶, andheart²⁷. BM-derived side population cells likely give rise to myocytesand vascular endothelium²⁸. This dye efflux technique may be affected bythe technique's inherent toxicity, causing viability after Hoechststaining and labeling to be 55% and even if analyzed immediatelystaining, viability is only 70%˜80%²⁹.

For the isolation of pure cell populations or for the expansion ofisolated cells for therapeutic use, expansion is often required.Multipotent adult progenitor cells (MAPCs)³⁰⁻³² from various organ andBM-derived multipotent stem cells (BMSCs) are clonally expanded,proliferate indefinitely without obvious genetic instability anddifferentiate into cells of all three embryonic germ layers. Someculturing techniques are difficult, require the use of large amounts ofanimal serum, require multiple expensive cytokines, and often timesrequire prolonged period to generate cell numbers that are sufficientfor clinical use. Any of these methods can be used for the isolation ofCD31⁺ multipotent stem cells.

Once isolated, CD31⁺ multipotent stem cells may be used for theprevention or treatment of virtually any disease characterized by anincrease in cell death, or a decrease in cell number. In particular, thepresent invention provides methods of treating disease and/or disordersor symptoms thereof which comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof the formulae herein to a subject (e.g., a mammal such as a human).Thus, one embodiment is a method of treating a subject suffering from orsusceptible to a cardiovascular disease, such as myocardial infarction,congestive heart failure, peripheral vascular obstructive disease, aperipheral neuropathy, such as diabetic, ischemic, toxic orchemical-induced neuropathies, an ischemic disease or disorder, stroke,cerebrovascular disease, liver failure, renal failure, diabetes, boneand joint diseases (osteoporosis, osteoarthritis), spinal cord injury,unhealed wound, skin gangrene or ulcer or any degenerative disease(e.g., muscular dystrophy, amyotrophic lateral sclerosis, diabetes,inflammatory bowel disease, rheumatoid arthritis, Parkinson's disease,Huntington's disease, Alzheimer's disease).

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa CD31 expressing multipotent stem cell described herein. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a disease, disorder, or symptomthereof. Determination of those subjects “at risk” can be made by anyobjective or subjective determination by a diagnostic test or opinion ofa subject or health care provider (e.g., genetic test, enzyme or proteinmarker, Marker (as defined herein), family history, and the like). Thecompounds herein may be also used in the treatment of any otherdisorders in which a need for the cells of the invention may beimplicated.

Endothelial Progenitor Cells

Endothelial progenitor cells exist in peripheral blood and bone marrow,and contribute to postnatal vasculogenesis, i.e., the de novodevelopment of vessels from stem or progenitor cells. Recently, thetherapeutic potential of bone marrow derived stem or progenitor cellshas been widely explored in various cardiovascular diseases.Collectively, studies have demonstrated that in both animal models andearly cohorts of patients, stem/progenitor cell therapy is safe andfeasible, and the clinical outcomes are promising. Mechanistically, inthese animal models, differentiation of endothelial progenitor cellsinto vasculature (vasculogenesis) has been considered as the majortherapeutic mechanism. Additionally, studies have demonstrated thatparacrine effects of endothelial progenitor cells can play a) crucialrole for mediating therapeutic effects. Endothelial progenitor cellscontain abundant and multiple cytokines, such as VEGF, IGF-1 and bFGFthat can function as angiogenic and neurotrophic factors. Also,endothelial progenitor cells are involved in disease pathogenesis.Decreased availability and impaired function of endothelial progenitorcells in diabetes may contribute to the development of diabeticcomplications including cardiomyopathy and peripheral vascular diseases,which are characterized by defective neovascularization.

Angiogenesis and vasculogenesis are responsible for the development ofthe vascular system in embryos. Vasculogenesis refers to the de novodevelopment of blood vessels from endothelial progenitor cells (EPCs) orangioblasts that differentiate into endothelial cells (ECs). Incontrast, angiogenesis refers to the formation of new vasculature frompreexisting blood vessels through proliferation, migration, andremodeling of fully differentiated ECs. The long held belief thatvasculogenesis occurs exclusively during development and that in theadult, new vessels are formed solely by angiogenesis, was dismantled bythe finding that circulating EPCs, isolated from adult species, coulddifferentiate along an EC lineage in vitro, providing evidence for theexistence of postnatal vasculogenesis. Initially, Flk-1 and CD34, sharedby angioblasts and hematopoietic cells were used to isolate putativeangioblasts from the mononuclear cell fraction of the peripheral blood(Asahara et al., Science. 1997; 275:964-967). Meanwhile, EPCssubsequently were isolated from umbilical cord blood, bone marrow (BM),and CD34⁺ or CD133⁺ hematopoietic stem cells (Asahara et al., Circ Res.Aug. 6, 1999; 85(3):221-228; Murohara et al., Journal of ClinicalInvestigation. 2000; 105:1527-1536; Shi et al., Blood. 1998; 92:362-367;Rafii et al., Journal of Clinical Investigation. 2000; 105:17-19). Thesecells differentiate into endothelial cells, as shown by expression ofvarious endothelial proteins (KDR, von Willebrand factor, endothelialnitric oxidase synthase (eNOS), VE-cadherin, CD146), uptake ofDiI-acetylated low-density lipoprotein (DiI-acLDL) and binding oflectin. In animal models of ischemia, heterologous, homologous, andautologous EPCs have been shown to incorporate into sites of activeneovascularization in ischemic tissue (Shi et al., Blood. 1998;92:362-367 Asahara et al., EMBO J. 1999; 18:3964-3972 Hatzopoulos etal., Development. 1998; 125(8):1457-1468; Niklason et al., Science.1999; 286:1493-1494; Rekhter et al., Circulation Research. 1998;83:705-713; Gerber et al., Development. 1999; 126:1149-1159; Gunsiliuset al., Lancet. 2000; 355:1688-1691)

As reported in more detail below, therapeutic intervention bytransplantation of CD31⁺ cells (or their progenitors or progeny)reversed or attenuated ischemic injury (e.g., hind limb ischemia andmyocardial infarction) by contributing to the vasculature of the damagedtissue.

Endothelial Cell Promoting Conditions

Once isolated, a CD31⁺ stem cell useful in the methods of the inventionmay be maintained indefinitely in culture. In one approach, isolatedCD31⁺ stem cells are used with or without expansion in vitro to increasethe number of cells suitable for therapeutic administration (e.g., cellshaving hemangioblastic activity or having the potential to differentiateinto an endothelial cell). Alternatively or subsequently, a CD31⁺ cellof the invention is incubated under conditions that promote endothelialcell differentiation. Examples of endothelial cell promoting conditionsare known in the art. See, for example, U.S. Pat. No. 5,980,887;PCT/US99/05130 (WO 99/45775) and references cited therein, hereinincorporated by reference. In one embodiment, the stem cells of theinvention are contacted with any one or more of the following factorsthat promote or support cardiomyogenic, neural or endothelial growth,proliferation, or cell differentiation: acidic and basic fibroblastgrowth factors (aFGF (GenBank Accession No. NP_(—)149127) and bFGF(GenBank Accession No. AAA52448)), vascular endothelial growth factor(VEGF-1, (GenBank Accession No. AAA35789 or NP_(—)001020539)), VEGF-2,VEGF165, epidermal growth factor (EGF)(GenBank Accession No.NP_(—)001954)), transforming growth factor α and β (TGF-α (GenBankAccession No. NP_(—)003227) and TFG-β (GenBank Accession No. 1109243A)),platelet-derived endothelial cell growth factor (PD-ECGF) (GenBankAccession No. NP_(—)001944)), platelet-derived growth factor (PDGF)(GenBank Accession No. 1109245A), tumor necrosis factor α (TNF-α)(GenBank Accession No. CAA26669), hepatocyte growth factor (HGF)(GenBank Accession No. BAA14348), insulin like growth factor (IGF)(GenBank Accession No. P08833), erythropoietin (GenBank Accession No.P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF)(GenBank Accession No. AAB59527) Sonic hedgehog (SHh, GenBank AccessionNo. NP_(—)000184), granulocyte/macrophage CSF (GM-CSF (GenBank AccessionNo. NP_(—)000749)), angiopoetin-1 (Ang1 (GenBank Accession No.NP_(—)001137)), angiopoietin-2 (Ang-2, GenBank Accession No.NP_(—)001138), stromal cell derived factor (GenBank Accession No.NP_(—)008854), hypoxia inducible factor (HIF-1 (GenBank Accession No.NP_(—)001521), thrombopoietin, and interleukin 2, interleukin 6, stemcell factor (SCF), FLT3, and nitric oxide synthase (NOS) (GenBankAccession No. AAA36365); and functional fragments thereof. See, alsoKlagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991); Folkman, etal., J. Biol. Chem., 267:10931-10934 (1992) and Symes, et al., CurrentOpinion in Lipidology, 5:305-312 (1994). Muteins or fragments of suchfactors may be used as long as they induce or promote formation ofendothelial cells. In one embodiment, an endothelial cell promotingcondition includes contact with VEGF, particularly VEGF-1, VEGF-2, andor VEGF165. Additionally preferred endothelial cell promoting conditionsinclude contact with certain cell matrix proteins, such as fibronectin.Preferred angiogenic factors and mitogens (and methods of use) aredisclosed herein as well as U.S. Pat. No. 5,980,887 and WO 99/45775.

Endothelial cells can be contacted with such angiogenic factors ormitogens prior to, during or following transplantation. Methods formaking and using EPCs have been disclosed. See U.S. Pat. No. 5,980,887,for example. Typical methods can include isolating the EPCs from themammal and contacting the EPCs with at least one angiogenic factorand/or mitogen ex vivo.

Administration

Compositions comprising a CD31⁺ cell having the potential todifferentiate into an endothelial cell (e.g., CD31⁺ multipotent stemcells or progenitors or progeny thereof) can be provided systemically orlocally for the treatment of a disease characterized by cell death orloss. In one embodiment, a CD31⁺ cell of the invention is providedlocally to a neural tissue (e.g., a sensory or motor neuron) for thetreatment of neuropathy. Alternatively, compositions comprising a CD31⁺expressing cell having hemangioblastic activity or having the potentialto differentiate into an endothelial cell (e.g., multipotent stem cells,endothelial progenitor cells, mesenchymal stem cells, mononuclear cells,or progenitors or progeny thereof) can be provided indirectly to theneural tissue of interest, for example, by local administration into amuscle comprising the neuron or into the circulatory system supplyingthe neuron. Following transplantation or implantation, the cells mayengraft and differentiate into endothelial cells. “Engraft” refers tothe process of cellular contact and incorporation into an existingtissue of interest in vivo. Expansion and differentiation agents can beprovided prior to, during or after administration to increase productionof endothelial cells in vivo.

Compositions of the invention include pharmaceutical compositionscomprising a CD31⁺ expressing cell having hemangioblastic activity,including having the potential to differentiate into a hematopoieticcell or an endothelial cell (e.g., multipotent stem cells, endothelialprogenitor cells, mesenchymal stem cells, mononuclear cells, orprogenitors or progeny thereof) and a pharmaceutically acceptablecarrier. Administration can be autologous or heterologous. For example,a CD31⁺ expressing cell having the potential to differentiate into anendothelial cell can be obtained from one subject, and administered tothe same subject or a different, compatible subject.

A CD31⁺ cell of the invention can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, intramuscular injection,intraneural injection or parenteral administration. When administering atherapeutic composition of the present invention (e.g., a pharmaceuticalcomposition), it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion).

Compositions of the invention can be conveniently provided as sterileliquid preparations, e.g., isotonic aqueous solutions, suspensions,emulsions, dispersions, or viscous compositions, which may be bufferedto a selected pH. Liquid preparations are normally easier to preparethan gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired. Such compositions may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose, dextrose, or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances such aswetting, dispersing, or emulsifying agents (e.g., methylcellulose), pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the multipotent stem cells, endothelial progenitorcells, mesenchymal stem cells, mononuclear cells, or progenitors orprogeny thereof.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents, such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount that will achieve the selectedviscosity. Obviously, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form).

A method to potentially increase cell survival when introducing thecells into a subject in need thereof is to incorporate CD31⁺ expressingmultipotent stem cells and their progenitor cells or their progeny(e.g., in vivo, ex vivo or in vitro derived) of interest into abiopolymer or synthetic polymer. Depending on the subject's condition,the site of injection might prove inhospitable for cell seeding andgrowth because of scarring or other impediments. Examples of biopolymerinclude, but are not limited to, cells mixed with fibronectin, fibrin,fibrinogen, thrombin, collagen, proteoglycans and other proteincomponents of the extracellular matrix. This could be constructed withor without included expansion factors, differentiation factors,endothelial cell promoting factors, neurotrophic factors, or angiogenicfactors. Additionally, these could be in suspension, but residence timeat sites subjected to flow would be nominal. Another alternative is athree-dimensional gel with cells entrapped within the interstices of thecell biopolymer admixture. Again, expansion or differentiation factorscould be included with the cells. These could be deployed by injectionvia various routes described herein.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the cells having the potential todifferentiate into an endothelial cell. This will present no problem tothose skilled in chemical and pharmaceutical principles, or problems canbe readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein. Additionally, these could be insuspension, but residence time at sites subjected to flow would benominal. Another alternative is a three-dimensional gel with cellsentrapped within the interstices of the cell biopolymer admixture.Again, expansion or differentiation factors could be included with thecells. These could be deployed by injection via various routes describedherein.

CD31⁺ expressing multipotent stem cells can be cultured, treated withagents and/or administered in the presence of polymer scaffolds. Polymerscaffolds are designed to optimize gas, nutrient, and waste exchange bydiffusion. Polymer scaffolds can comprise, for example, a porous,non-woven array of fibers. The polymer scaffold can be shaped tomaximize surface area, to allow adequate diffusion of nutrients andgrowth factors to the cells. Taking these parameters into consideration,one of skill in the art could configure a polymer scaffold havingsufficient surface area for the cells to be nourished by diffusion untilnew blood vessels interdigitate the implanted engineered-tissue usingmethods known in the art. Polymer scaffolds can comprise a fibrillarstructure. The fibers can be round, scalloped, flattened, star-shaped,solitary or entwined with other fibers. Branching fibers can be used,increasing surface area proportionately to volume.

Unless otherwise specified, the term “polymer” includes polymers andmonomers that can be polymerized or adhered to form an integral unit.The polymer can be non-biodegradable or biodegradable, typically viahydrolysis or enzymatic cleavage. The term “biodegradable” refers tomaterials that are bioresorbable and/or degrade and/or break down bymechanical degradation upon interaction with a physiological environmentinto components that are metabolizable or excretable, over a period oftime from minutes to three years, preferably less than one year, whilemaintaining the requisite structural integrity. As used in reference topolymers, the term “degrade” refers to cleavage of the polymer chain,such that the molecular weight stays approximately constant at theoligomer level and particles of polymer remain following degradation.

Materials suitable for polymer scaffold fabrication include polylacticacid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA),polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA),polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, modified cellulose, collagen, polyhydroxybutyrate,polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid),polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyaminoacids, polyorthoesters, polyacetals, polycyanoacrylates, degradableurethanes, aliphatic polyester polyacrylates, polymethacrylate, acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinylimidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinylalcohol, Teflon®, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(ε-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989).

Factors, including but not limited to nutrients, growth factors,inducers of differentiation or de-differentiation, products ofsecretion, immunomodulators, cytokines, neurotrophic factors, myogenicfactors, angiogenic factors, inhibitors of inflammation, regressionfactors, hormones, various tissue extracts (e.g. from myocardium orskeletal muscle) or other biologically active compounds can beincorporated into or can be provided in conjunction with the polymerscaffold.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the multipotent stem cells,endothelial progenitor cells, mesenchymal stem cells, and theirprogenitor cells as described in the present invention.

One consideration concerning the therapeutic use of multipotent stemcells, hematopoietic stem cells, endothelial progenitor cells,mesenchymal stem cells, and their progenitor cells of the invention isthe quantity of cells necessary to achieve an optimal effect. In currenthuman studies of autologous mononuclear peripheral blood cells,empirical doses ranging from 1 to 4×10⁷ cells have been used withencouraging results. The methods of the invention may requireoptimization of the amount of cells injected into a tissue of interest.Thus, the quantity of cells to be administered will vary depending onthe neural tissue or the subject being treated. In one embodiment,between 10⁴ to 10⁸, 10⁶ to 10⁸, or 10⁵ to 10⁹ cells are implanted. Inother embodiments, 10⁵ to 10⁷ cells are implanted. In still otherembodiments, 3×10⁷ stem cells of the invention can be administered to ahuman subject. The precise determination of an effective dose may bebased on factors individual to each patient, including their size, age,sex, weight, and condition. Therefore, dosages are determinedempirically using no more than routine by those skilled in the art fromthis disclosure and the knowledge in the art.

CD31⁺ expressing multipotent stem cells and progenitor cells of theinvention can comprise a purified population of CD31⁺ expressing stemcells having hemangioblastic activity or having the potential todifferentiate into an endothelial cell and other lineages. Those skilledin the art can readily determine the percentage of such cells in apopulation using various well-known methods, such as fluorescenceactivated cell sorting (FACS). Desirable ranges of purity in mixedpopulations comprising multipotent stem cells, endothelial progenitorcells, mesenchymal stem cells, or progenitor cells of the inventioncells are about 50 to about 55%, about 55 to about 60%, and about 65 toabout 70%. More desirably, the purity is about 70 to about 75%, about 75to about 80%, about 80 to about 85%; and still more desirably the purityis about 85 to about 90%, about 90 to about 95%, and about 95 to about100%. Purity of CD31⁺ expressing multipotent stem cells can bedetermined according to the marker profile within a population. Dosagescan be readily adjusted by those skilled in the art (e.g., a decrease inpurity may require an increase in dosage).

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or agent(s)) are present in anamount of 0.001 to 50% (weight) solution in phosphate buffered saline,and the active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10wt %, and still more preferably about 0.05 to about 5 wt %. Of course,for any composition to be administered to an animal or human, and forany particular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD₅₀ in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

The number of CD31⁺ expressing multipotent stem cells of the inventioncan be increased by increasing the survival or proliferation of existingstem cells, or their progenitor cells.

Agents (e.g., expansion agents) which increase proliferation or survivalof a CD31⁺ expressing cell or progenitors or progeny thereof are usefulaccording to the invention. Agents comprising growth factors are knownin the art to increase proliferation or survival of stem cells. Forexample, U.S. Pat. Nos. 5,750,376 and 5,851,832 describe methods for thein vitro culture and proliferation of stem cells using TGF. An activerole in the expansion and proliferation of stem cells has also beendescribed for BMPs (Zhu, G et al, (1999) Dev. Biol. 215: 118-29 andKawase, E. et al, (2001) Development 131: 1365) and Wnt proteins(Pazianos, G et al, (2003) Biotechniques 35: 1240 and Constantinescu, S.(2003) J. Cell Mol. Med. 7: 103). U.S. Pat. Nos. 5,453,357 and 5,851,832describe proliferative stem cell culture systems that utilize FGFs. Thecontents of each of these references are specifically incorporatedherein by reference for their description of expansion agents known inthe art.

Agents comprising growth factors are also known in the art to increasemobilization of stem cells from the bone marrow into the peripheralblood. Mobilizing agents include but are not limited to GCSF or GMCSF.An agent that increases mobilization of stem cells into the blood can beprovided before peripheral blood harvest or alternatively, to augment orsupplement other methods of the invention where it would be desirable toincrease circulating levels of stem cells (e.g., to increase targetingof the cells to the neural tissue).

Agents comprising cell-signaling molecules are also known in the art toincrease proliferation or survival of stem cells. For example, U.S.Patent Application No. 20030113913 describes the use of retinoic acid instem cell self renewal in culture. The contents of each of thesereferences are specifically incorporated herein by reference for theirdescription of expansion agents known in the art.

Agents comprising pharmacological or pharmaceutical compounds are alsoknown in the art to increase production or survival of stem cells.

Agents comprising signaling molecules are also known to inducedifferentiation of endothelial cells. The contents of each of thesereferences are specifically incorporated herein by reference for theirdescription of differentiation agents known in the art.

Agents comprising pharmacological or pharmaceutical compounds are alsoknown in the art to induce differentiation of stem cells.

Agents can be provided directly to an ischemic tissue or to a neuraltissue effected by a neuropathy (e.g., motor neuron, sensory neuron).Alternatively, agents can be provided indirectly to the neural tissue ofinterest, for example, by local administration into the circulatorysystem or into the muscle that comprises the neural tissue.

Agents can be administered to subjects in need thereof by a variety ofadministration routes. Methods of administration, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,intraocular, buccal, intracisternal, intracerebroventricular,intratracheal, nasal, transdermal, within/on implants, e.g., fibers suchas collagen, osmotic pumps, or grafts comprising appropriatelytransformed cells, etc., or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, intramuscular, intraperitoneal, orinfusion. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis. A particular method ofadministration involves coating, embedding or derivatizing fibers, suchas collagen fibers, protein polymers, etc. with therapeutic proteins.Other useful approaches are described in Otto, D. et al., J. Neurosci.Res. 22: 83 and in Otto, D. and Unsicker, K. J. Neurosci. 10: 1912.

In vitro and ex vivo applications can involve culture of the multipotentstem cells, endothelial progenitor cells, mesenchymal stem cells, andprogenitor cells with the selected agent to achieve the desired result.For example, cultures of cells (from the same individual and fromdifferent individuals) can be treated with expansion agents to increasethe number of cells of interest. Alternatively, the cultures are treatedwith differentiation agents of interest to stimulate the production ofcells having the desired characteristics. Cells produced by thesemethods can then be used for a variety of therapeutic applications(e.g., localized implantation).

Multipotent stem cells, endothelial progenitor cells, mesenchymal stemcells, and progenitor cells derived from cultures of the invention canbe implanted into a host. The transplantation can be autologous, suchthat the donor of the stem cells is also the recipient of the stemcells. The transplantation can be heterologous, such that the donor ofthe stem cells is not the recipient of the stem cells. In oneembodiment, once transferred into a host, the cells engraft in themicrovasculature of the host neural tissue.

Agents of the invention may be supplied along with additional reagentsin a kit. The kits can include instructions for the treatment regime orassay, reagents, equipment (test tubes, reaction vessels, needles,syringes, etc.) and standards for calibrating or conducting thetreatment or assay. The instructions provided in a kit according to theinvention may be directed to suitable operational parameters in the formof a label or a separate insert. Optionally, the kit may furthercomprise a standard or control information so that the test sample canbe compared with the control information standard to determine whether aconsistent result is achieved.

Genetically Modified Stem Cells

Prior to administration, CD31⁺ multipotent stem cells, their progenitorsor their progeny, described herein can optionally be geneticallymodified, in vitro, in vivo or ex vivo, by introducing heterologous DNAor RNA or protein into the cell by a variety of recombinant methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adeno-associated virus, adenovirus, Sindbisvirus, and bovine papillomavirus, for example; (2) chemical transfer,including calcium phosphate transfection and DEAE dextran transfectionmethods; (3) membrane fusion transfer, using DNA-loaded membranousvesicles such as liposomes, red blood cell ghosts, and protoplasts, forexample; and (4) physical transfer techniques, such as microinjection,electroporation, or direct “naked” DNA transfer.

The CD31⁺ stem cells of the invention, their progenitors or theirprogeny, can be genetically altered by insertion of pre-selectedisolated DNA, by substitution of a segment of the cellular genome withpre-selected isolated DNA, or by deletion of or inactivation of at leasta portion of the cellular genome of the cell. Deletion or inactivationof at least a portion of the cellular genome can be accomplished by avariety of means, including but not limited to genetic recombination, byantisense technology (which can include the use of peptide nucleicacids, or PNAs), or by ribozyme technology, for example. The alteredgenome may contain the genetic sequence of a selectable or screenablemarker gene that is expressed so that the cell with altered genome, orits progeny, can be differentiated from cells having an unalteredgenome. For example, the marker may be a green, red, yellow fluorescentprotein, β-galactosidase, the neomycin resistance gene, a geneticallyaltered stem cell, or its progeny, may contain DNA encoding atherapeutic protein (e.g., a protein that increases angiogenesis,increases endothelial cell or Schwann cell proliferation, or decreasesapoptosis) under the control of a promoter that directs strongexpression of the recombinant protein. Alternatively, the cell mayexpress a gene that can be regulated by an inducible promoter or othercontrol mechanism where conditions necessitate highly controlledregulation or timing of the expression of a protein, enzyme, or othercell product. Such CD31⁺ stem cells, when transplanted into a subjectsuffering from a disease, for example, including but not limited tomyocardial infarction, congestive heart failure, peripheral vascularobstructive disease, ischemia, limb ischemia, stroke, transientischemia, reperfusion injury, peripheral neuropathy, diabeticneuropathy, toxic neuropathy, diabetic dementia, or autonomicneuropathy, spinal cord injury, leukemia, lymphoma, myelodysplasticsyndrome, pancytopenia, anemia, thrombocytopenia, leukopenia, liverfailure, renal failure, diabetes, rheumatoid arthritis, osteoarthritis,skin wound, diabetic foot or ulcer, gangrene, diabetic wound andosteoporosis, confer a therapeutic benefit.

Proteins expressed in genetically modified cells include any proteincapable of supporting or enhancing tissue repair, tissue regeneration,neural function or angiogenesis. Such proteins include angiogeniccytokines and neurotrophic factors. In one embodiment, the CD31⁺ stemcell of the invention, its progenitor or its progeny, expressheterologous DNA encoding a polypeptide or fragment thereof that encodesa therapeutic polypeptide (e.g., acidic and basic fibroblast growthfactors, vascular endothelial growth factor, VEGF-2, VEGF165, epidermalgrowth factor, transforming growth factor α and β, platelet-derivedendothelial growth factor, platelet-derived growth factor A, B, E, tumornecrosis factor α, hepatocyte growth factor, insulin like growth factor1, and 2, erythropoietin, colony stimulating factor, macrophage-CSF,Sonic hedgehog, granulocyte/macrophage CSF, angiopoetin-1,angiopoietin-2, stromal cell derived factor (SDF-1) Hypoxia induciblefactor (HIF-1) and nitric oxide synthase). Insertion of one or morepre-selected DNA sequences can be accomplished by homologousrecombination or by viral integration into the host cell genome. Thedesired gene sequence can also be incorporated into the cell,particularly into its nucleus, using a plasmid expression vector and anuclear localization sequence. Methods for directing polynucleotides tothe nucleus have been described in the art. Calcium phosphatetransfection can be used to introduce plasmid DNA containing a targetgene or polynucleotide into isolated or cultured stem cells or theirprogenitors and is a standard method of DNA transfer to those of skillin the art. DEAE-dextran transfection, which is also known to those ofskill in the art, may be preferred over calcium phosphate transfectionwhere transient transfection is desired, as it is often more efficient.Since the cells of the present invention are isolated cells,microinjection can be particularly effective for transferring geneticmaterial into the cells. This method is advantageous because it providesdelivery of the desired genetic material directly to the nucleus,avoiding both cytoplasmic and lysosomal degradation of the injectedpolynucleotide. This technique has been used effectively to accomplishperipheral blood derived modification in transgenic animals. Cells ofthe present invention can also be genetically modified usingelectroporation.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, which form a stable complex with thepolynucleotide. For stabilization of the liposome complex, dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPA)can be added. Commercially available reagents for liposomal transferinclude Lipofectin (Life Technologies). Lipofectin, for example, is amixture of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N—N—N-trimethyl ammonia chloride and DOPE.Liposomes can carry larger pieces of DNA, can generally protect thepolynucleotide from degradation, and can be targeted to specific cellsor tissues. Cationic lipid-mediated gene transfer efficiency can beenhanced by incorporating purified viral or cellular envelopecomponents, such as the purified G glycoprotein of the vesicularstomatitis virus envelope (VSV-G). Gene transfer techniques which havebeen shown effective for delivery of DNA into primary and establishedmammalian cell lines using lipopolyamine-coated DNA can be used tointroduce target DNA into the stem cells described herein.

Naked plasmid DNA can be injected directly into a tissue mass formed ofcells from the isolated peripheral blood or their progenitors. Thistechnique has been shown to be effective in transferring plasmid DNA toskeletal muscle tissue, where expression in mouse skeletal muscle hasbeen observed for more than 19 months following a single intramuscularinjection. More rapidly dividing cells take up naked plasmid DNA moreefficiently. Therefore, it is advantageous to stimulate cell divisionprior to treatment with plasmid DNA. Microprojectile gene transfer canalso be used to transfer genes into stem cells either in vitro or invivo. The basic procedure for microprojectile gene transfer wasdescribed by J. Wolff in Gene Therapeutics (1994), page 195. Similarly,microparticle injection techniques have been described previously, andmethods are known to those of skill in the art. Signal peptides can bealso attached to plasmid DNA to direct the DNA to the nucleus for moreefficient expression.

Viral vectors are used to genetically alter stem cells of the presentinvention and their progeny. Viral vectors are used, as are the physicalmethods previously described, to deliver one or more target genes,polynucleotides, antisense molecules, or ribozyme sequences, forexample, into the cells. Viral vectors and methods for using them todeliver DNA to cells are well known to those of skill in the art.Examples of viral vectors that can be used to genetically alter thecells of the present invention include, but are not limited to,adenoviral vectors, adeno-associated viral vectors, retroviral vectors(including lentiviral vectors), alphaviral vectors (e.g., Sindbisvectors), and herpes virus vectors.

Screening Assays

The invention provides methods for identifying modulators, i.e.,candidate or test compounds or agents (e.g., proteins, peptides,peptidomimetics, peptoids, polynucleotides, small molecules or otherdrugs) that are useful for the treatment of neuropathy. Agents thusidentified can be used to increase, for example, proliferation,survival, engraftment, or differentiation of a stem cell or itsprogenitor e.g., in a therapeutic protocol. In one embodiment, the agentmodulates a cell of the invention thereby enhancing angiogenesis in aneural tissue of interest.

The test agents of the present invention can be obtained singly or usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; peptoid libraries (librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann, R. N. (1994)et al., J. Med. Chem. 37:2678-85); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992), Biotechniques 13:412-421), or on beads (Lam (1991), Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

Chemical compounds to be used as test agents (i.e., potential inhibitor,antagonist, agonist) can be obtained from commercial sources or can besynthesized from readily available starting materials using standardsynthetic techniques and methodologies known to those of ordinary skillin the art. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds identified by the methods described herein are known in theart and include, for example, those such as described in R. Larock(1989) Comprehensive Organic Transformations, VCH Publishers; T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nded., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Combinations of substituents and variables in compounds envisioned bythis invention are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein. (e.g., transport, storage,assaying, therapeutic administration to a subject).

The compounds described herein can contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The compounds described herein can also berepresented in multiple tautomeric forms, all of which are includedherein. The compounds can also occur in cis- or trans- or E- or Z-doublebond isomeric forms. All such isomeric forms of such compounds areexpressly included in the present invention.

Test agents of the invention can also be peptides (e.g., growth factors,cytokines, receptor ligands) or polynucleotides encoding such peptides.

Screening methods of the invention identify agents that enhance orinhibit a biological activity of a cell of the invention. In oneembodiment, a cell of the invention (e.g., multipotent stem cell,endothelial progenitor cell, mesenchymal stem cell, or other progenitorcell) is contacted with the agent prior to implantation in a host. Inanother embodiment, an agent is administered in combination with a cellof the invention. Desirably, the agent increases angiogenesis in aneural tissue of interest, increases Schwann cell proliferation,increases endothelial cell proliferation, increases neural conductance,increases pain responsiveness, decreases apoptosis, or is otherwiseuseful for the treatment of a diabetic neuropathy.

In one embodiment, a CD31⁺ cells of the invention is contacted with theagent in vitro prior to implantation in a host. The treated cell is thenlocally delivered to a tissue of interest. The biological function orvascularity of the tissue is compared between a host that received thetreated cell relative to a host that received an untreated control cell.An increase in the biological function or vascularity of the tissue thatreceived the treated cell identifies the agent as useful in the methodsof the invention.

In another embodiment, an agent is locally administered to a tissue ofinterest in combination (e.g., prior to, during, or following)implantation of a cell of the invention. The biological function orvascularity of the tissue contacted with the agent is compared to thebiological function of in a control host that did not receive the agent.An increase in the biological function or vascularity of the tissuecontacted with the combination identifies the agent as useful in themethods of the invention.

In practicing the methods of the invention, it may be desirable toemploy a purified population of CD31⁺ multipotent stem cells,endothelial progenitor cells, mesenchymal stem cells, and progenitorcells. A purified population of multipotent stem cells, endothelialprogenitor cells, mesenchymal stem cells, and progenitor cells has about50-55%, 55-60%, 60-65% and 65-70% purity. In other embodiments, thepurity is about 70-75%, 75-80%, 80-85%; and in still other embodimentsthe purity is about 85-90%, 90-95%, and 95-100%.

Agents useful in the methods of the invention can also be detected byidentifying an increase in expression of a cytokine or other desirablemarker. The level of expression can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the geneticmarkers; measuring the amount of protein encoded by the genetic markers;or measuring the activity of the protein encoded by the genetic markers.

The level of mRNA corresponding to a marker can be determined both by insitu and by in vitro formats. The isolated mRNA can be used inhybridization or amplification assays that include, but are not limitedto, Southern or Northern analyses, polymerase chain reaction analysesand probe arrays. In one format, mRNA (or cDNA) is immobilized on asurface and contacted with the probes, for example by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In an alternative format, theprobes are immobilized on a surface and the mRNA (or cDNA) is contactedwith the probes, for example, in a two-dimensional gene chip arraydescribed below. A skilled artisan can adapt known mRNA detectionmethods for use in detecting the level of mRNA encoded by the markersdescribed herein.

The level of mRNA in a sample can be evaluated with nucleic acidamplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202),ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA88:189-193), self sustained sequence replication (Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplificationsystem (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177),Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rollingcircle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques known in the art. As usedherein, amplification primers are defined as being a pair of nucleicacid molecules that can anneal to 5′ or 3′ regions of a gene (plus andminus strands, respectively, or vice-versa) and contain a short regionin between. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes themarker being analyzed.

Assays for Angiogenesis, Proliferation, and Apoptosis

The invention provides methods for increasing angiogenesis in a tissueof interest, increasing endothelial cell proliferation, decreasingapoptosis, or is otherwise useful for the treatment of a diabeticneuropathy. Methods for measuring an increase in angiogenesis are alsoknown in the art and are described herein. In general, angiogenesis canbe assayed by measuring the number of non-branching blood vesselsegments (number of segments per unit area), the functional vasculardensity (total length of perfused blood vessel per unit area), thevessel diameter, or the vessel volume density (total of calculated bloodvessel volume based on length and diameter of each segment per unitarea). Angiogenesis can also be quantitated using endothelial cellmarkers. For example, angiogenesis can be assayed in a cardiac, skeletalor neural tissue using immunohistochemical staining with antibodiesprepared against a specific endothelial cell marker isolectin B4 (VectorLaboratories). Capillary density is evaluated morphometrically byhistological examination of randomly selected fields of tissue sections.Capillaries are recognized as tubular structures positive for isolectin.Such methods are described, for example, by Iwakura et al., Circulation2003; 108: 3115-21.

Methods of assaying cell growth and proliferation are known in the art.See, for example, Kittler et al. (Nature. 432 (7020):1036-40, 2004) andMiyamoto et al. (Nature 416(6883):865-9, 2002). Assays for cellproliferation generally involve the measurement of DNA synthesis duringcell replication. In one embodiment, DNA synthesis is detected usinglabeled DNA precursors, such as ([³H]-Thymidine or5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals)and then the incorporation of these precursors into genomic DNA duringthe S phase of the cell cycle (replication) is detected (Ruefli-Brasseet al., Science 302(5650):1581-4, 2003; Gu et al., Science 302(5644):445-9, 2003).

Assays for measuring cell survival are known in the art, and aredescribed, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8);Kangas et al. (Med. Biol. 62, 338-43, 1984); Lundin et al., (Meth.Enzymol. 133, 27-42, 1986); Petty et al. (Comparison of J. Biolum.Chemilum. 10, 29-34, 1995); and Cree et al. (AntiCancer Drugs 6:398-404, 1995). Cell viability can be assayed using a variety ofmethods, including MTT(3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop,Bioorg. & Med. Chem. Lett. 1: 611, 1991; Cory et al., Cancer Comm. 3,207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays forcell viability are also available commercially. These assays include butare not limited to CELLTITER-GLO® Luminescent Cell Viability Assay(Promega), which uses luciferase technology to detect ATP and quantifythe health or number of cells in culture, and the CellTiter-Glo®Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH)cytotoxicity assay (Promega).

Kits

The invention provides kits for the treatment or prevention of ischemia,related tissue damage, or any other disease characterized by increasedcell death or reduced cell number. In one embodiment, the kit includes atherapeutic or prophylactic composition containing an effective amountof CD31⁺ cells in unit dosage form. In some embodiments, the kitcomprises a sterile container which contains a therapeutic orprophylactic vaccine; such containers can be boxes, ampules, bottles,vials, tubes, bags, pouches, blister-packs, or other suitable containerforms known in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

If desired CD31⁺ cells of the invention are provided together withinstructions for administering it to a subject having or at risk ofdeveloping ischemia, related tissue damage, or any other diseasecharacterized by increased cell death or reduced cell number. Theinstructions will generally include information about the use of thecomposition for the treatment or prevention of ischemia or for enhancingangiogenesis to a tissue in need thereof. In other embodiments, theinstructions include at least one of the following: description of theexpression vector; dosage schedule and administration for treatment orprevention of ischemia or symptoms thereof; precautions; warnings;indications; counter-indications; overdosage information; adversereactions; animal pharmacology; clinical studies; and/or references. Theinstructions may be printed directly on the container (when present), oras a label applied to the container, or as a separate sheet, pamphlet,card, or folder supplied in or with the container.

Combination Therapies

Compositions and methods of the invention may be used in combinationwith any conventional therapy for ischemia, related tissue damage, orany other disease characterized by increased cell death or reduced cellnumber known in the art or in combination with any therapy known toincrease angiogenesis. In one embodiment, a CD31⁺ multipotent stem cellmay be used in combination with any pro-angiogenic therapy known in theart.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 CD31 is a Common Marker for Various Stem Cells

CD31 was identified as a surface epitope common to most stem orprogenitor cells that have therapeutic effects in cardiovascularregeneration or repair. In particular, FACS analysis demonstrated thatCD34⁺ human hematopoietic cells and precursors of both myeloid andlymphoid cells express high levels of CD31^(36, 37). 99% ofc-kit⁺sca-1⁺lineage⁻ cells (KSL cells), a population of BM-derivedhematopoietic stem cells, also expressed CD31³⁸. Side population cellsin BM are predominantly CD31 positive²⁹. In addition to adult stemcells, pluripotent embryonic stem cells also express CD31 constitutivelyat undifferentiated stages despite lacking vascular structure and theabsence of angiogenic factors³⁹. An analysis of the antigen expressionprofiles of BM-MNCs demonstrates that around 30% of whole BM-MNCsexpress the CD31 antigen (FIG. 1A) and CD31+ cells are co-expressed withother well-known stem cell markers such as CD34, c-kit and Flk-1 (FIG.1B). When lineage positive cells were depleted, these co-expressionpatterns were much more obvious (FIG. 1C). Taken together, thesefindings indicate that the CD31 antigen may function as a unique andcomprehensive marker that is expressed by most if not all stem cellsshowing hemangioblastic activity in the BM (FIG. 2).

Example 2 Harvesting BM-Mononuclear Cells and CD31 Selection

BM cells were harvested from mice after euthanasia. BM cells wereobtained by grinding thigh bones, forelimb bones, and sternum with PBS/2mM EDTA. After passing the lysate through 70 and 40 μm nylon filters toremove particulate debris, mononuclear cells were isolated by densitygradient centrifugation with Histopaque 1083 (Sigma). BM-MNCs werelabeled with rat anti-mouse-CD31 monoclonal antibody (BD Pharmingen) for30 minutes, followed by incubation with goat anti-rat IgG magnetic beads(Mitenyi Biotec) for 20 minutes. Then, the cells were sorted through amagnetic column (MACS®, Mitenyi Biotec). CD31⁻ cells pass through themagnetic column and the remaining cells in the column are CD31⁺ cells.Bead-bound cells (CD31⁺) are retrieved from the column by flushing withPBS. Results for sorting efficiency are shown in FIG. 3. Overall, atleast 85% purity was confirmed by flow cytometry after cell sorting withthe magnetic beads. Trypan blue staining revealed that CD31⁺ and CD31⁻cells were more than 90% viable after the procedure.

Example 3 Colony Forming Unit Assay

To investigate whether CD31⁺ or CD31⁻ cells have a higher potential forproliferation and differentiation to form mesodermal derivatives, colonyforming unit assays will be performed after lineage-committed celldepletion. An initial density of 7,000 cells/mL will be cultured onmethylcellulose media (Methocult GF M3534 or M3231 media, Stem CellTechnologies) and analyzed for colony formation after 10 days inculture^(27, 51, 52). Whole lineage-depleted BM-MNCs will serve as aninternal control for the colony forming culture.

Example 4 3. CD31⁺ Cells Express Multiple Angiogenic Genes

Gene expression profiles were assayed using reversetranscriptase-polymerase chain reaction (RT-PCR) and microarrayanalysis. To evaluate embryonic gene expression, RNA was extracted fromwhole BM-MNCs, CD31⁺ and CD31⁻ cells as previously described³³. cDNA wasgenerated using a reverse transcription kit (Promega) according to themanufacturer's instructions. PCR was performed on cDNA using mousespecific primers: octamer-binding transcription factor 4 (Oct4), RNAexonuclease 4 (Rex4), Nanog, Sox2, SSEA-1, and GAPDH. This analysisshowed that CD31+ cells express several embryonic genes exclusively(FIG. 4). Mouse embryonic stem cells (mES) serve as a positive controland mesenchymal stem cells (MSC) as a negative control. For theevaluation of the other genes known to be important for theidentification, growth and differentiation of mouse stem cellsmicroarray (GEArray S Series, Mouse Stem Cell Gene Array:MM-601.2)^(53, 54) examination is performed using RNA samples.

Example 5 CD31⁺ Cells Possess BM Repopulating Potential

To demonstrate that BM-derived CD31⁺ cells have self-renewing potential,BM repopulating potential was examined using a BM transplantation (BMT)model. GFP transgenic mice with a C57BL/6J background (male, 6-8 weeks)were used as donors for the BMT. C57BL/6J mice (female, 6-8 weeks,Jackson Laboratories), which were used as recipients for BMT. Theprocedure of BMT was performed as described previously⁵⁵. Briefly,recipient mice were lethally irradiated with 1,200 cGγ in 2 equal dosesof 600 cGγ delivered 3 hours apart. CD31⁺ or CD31⁻ cells were injectedintravenously into the tail vein. Recipients received antibiotic waterfor 1 month after transplantation and survival was monitored daily. At 6to 8 weeks after BMT, which is sufficient time for the reconstitution ofthe BM of the recipient mice, both BM and peripheral blood mononuclearcells were analyzed for the engraftment of GFP cells using FACS. Also,to confirm the long-term repopulation and engraftment, BM cells fromprimary recipients were harvested to isolate GFP+ cells, which were thentransplanted in secondary recipients. These studies showed that CD31⁺cells repopulated BM in lethally irradiated BMT models (FIG. 5). CD31⁺cells rescued lethally-irradiated recipients.

A fraction of CD31⁺ cells of BM mononuclear cells in C57BL/6 miceco-expressed well known stem cell markers including c-kit, Sca-1, andflk-1. The expression levels of these markers were distinct whenhematopoietic lineage positive cells were depleted (Lin⁻) from CD31⁺cells. Moreover, Lin⁻CD31⁺ cells exclusively expressed genescharacteristic of cells having pluripotency, including Oct4, Rex4,Nanog, and SSEA-1. A microarray analysis revealed that CD31⁺ cellsexpressed multiple angiogenic genes compared to CD31⁻ cells. Inparticular, only a CD31⁺ but not CD31⁻ fraction gave rise to endothelialprogenitor cells (EPCs) in a culture assay. To determine in vivoactivity, we performed BM repopulating experiments. All mice that weretransplanted with 1×10⁵ CD31⁺ cells survived after lethal irradiation,whereas mice that received CD31⁻ cells all died within 4 weeks (n=10,each).

FACS analysis is performed using a modification of previously describedprocedures^(33, 56, 57) to characterize subsets of CD31⁺ cells. Thisstudy uses the following exemplary antibodies (all are monoclonalantibody for mice and from BD Pharmingen): fluorescein isothiocyanate(FITC)-conjugated anti-CD31; phycoerythrin (PE)-conjugated anti-CD3,anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-CD45R (B220), anti-GR-1(Ly-6G), anti-Ter119, anti-CD45, anti-CD34, anti-c-kit, anti-Sca-1, andanti-Flk1. Furthermore, the surface antigens of lineage-depleted cellsis analysed: stem cells lineages (c-kit, Sca-1, CD34), vascularprogenitors (Flk-1), and embryonic stem cells (SSEA-1, Oct-4). Flowcytometry analysis is performed on a FACSCAN (Becton-Dickinson) or MoFlo(DAKO). Acquired data are analyzed using specialized software FlowJo®(Tree Star, Inc), version 5.7. Depletion of committed cells is performedas described below. Harvested BM-MNCs are incubated with a lineagecocktail of biotin-conjugated anti-mouse monoclonal antibodies (all fromMiltenyi Biotec): CD5, CD45R, CD11b, GR-1, 7-4 and Ter119. Magneticbeads are conjugated to a monoclonal anti-biotin antibody (MiltenyiBiotec). The antibody non-bound (lineage-depleted) fraction is collectedby negative selection from exposure to a magnetic filed.

Example 6 CD31+Cells Express Multiple Angiogenic Genes

Angiogenic gene expression was compared by microarray analysis ofBM-derived CD31+ and CD31− subpopulations. (GEArray®, Mouse AngiogenesisMicroarray, OMM-024, SuperArray)^(62, 63). RNA was extracted from wholeBM-MNCs, CD31⁺ and CD31⁻ cells. This array was designed to profile theexpression of 113 key genes involved in modulating the biologicalprocesses of angiogenesis; the array contains growth factors andreceptors, cytokines, chemokines, adhesion molecules, proteases,inhibitors and other matrix proteins, transcription factors, and others.The data were analyzed by GEArray Analysis Suite®. Results usingmicroarray analysis showed different patterns of gene expressionpatterns between CD31⁺ and CD31⁻ cells. (FIG. 6).

CD31+/CD31− Gene Expression Ratio

Ang-1 1.72-fold

Ang-2 2.55-fold

Akt 5.05-fold

Edg1 16.23-fold

bFGF 2.23-fold

IGF-1 2.01-fold

Example 7 Proliferation, Survival and Migration Assay Characterization

1×10⁴ cells of lineage-depleted CD31⁺ or CD31⁻ cells, respectively areseeded in each well of a 96-well plate in a final volume of 200 μl/well.After a 48 hour incubation, the cell survival reagent, WST-1 (Rochemolecular biochemicals) is added as 20 μL/well. The proliferativeactivities of each cell type is assessed by ELISAs for BrdUincorporation (Roche Diagnostics, Germany) after 24 and 72 hours. Themodified Boyden's chamber method is used for migration assays aspreviously described⁶⁴.

Example 8 Characterization of CD31+ Cells Angiogenic Potential

In Vitro Incorporation into HUVEC Monolayer.

Human umbilical vein endothelial cells (HUVECs, Cambrex) are cultured toform a monolayer in 6-well plates. One hundred thousand CD31⁺ or CD31⁻cells derived from GFP mice will be seeded onto a HUVEC monolayer. After4 hours of incubation, the wells are gently washed with PBS three timesand five random fields are selected to count the number of cellsincorporating into the HUVEC monolayer per unit area.

In Vitro Tube Formation.

Basement membrane matrix, Matrigel® (Becton Dickinson Labware) is addedto chamber slides. After one-hour incubation at room temperature, 2×10⁴cells are added to the chamber slides with 500 μl EGM-2 media. Twelvehours later, four representative fields are counted and the average ofthe total area of complete tubes formed by cells per unit area iscompared by Image-Pro Plus®.

Measurement of Cytokine Concentration of Supernatant.

To investigate whether CD31⁺ or CD31⁻ cells produce angiogenic,anti-apoptotic, mitogenic, and chemotactic factors, VEGF, HGF, IGF-1,bFGF, Angiopoietin-1, 2, TNF-α, IL-1β, IL-6, IL-8, IL-12, IL-16, andSDF-1 levels are measured in conditioned medium obtained from the CD31⁺or CD31⁻ 24 hours after medium replacement by ELISAs.

Example 9 CD31⁺ Cells Give Rise to Endothelial Progenitor Cells

CD31+ or CD31⁻ cells were plated onto a 2-well chamber slide, 2×10⁶cells/slide density. The cells were cultured in culture media, 5% fetalbovine serum (FBS)/endothelial basal media (EBM-2) medium supplementedwith 5% fetal bovine serum, antibiotics, and cytokine cocktail(SingleQuots) (Clonetics, San Diego, Calif.) consisting of humanepidermal growth factor, vascular endothelial growth factor (VEGF),human fibroblast growth factor-basic (FGF-2), insulin-like growthfactor-1 (IGF-1), ascorbic acid. (SingleQuots, Clonetics). At day 4,attached cells were evaluated by staining. Triple positive cells showingDiI-acetylated low density lipid (acLDL) (Molecular Probe) uptake,FITC-conjugated BS-1 lectin (Bandeiraea simplicifolia lectin I, VectorLaboratories) binding and DAPI positivity in nucleus were counted asEPCs as previously described^(7, 65). At least 5 randomly chosen fieldswere averaged for statistical analysis. Culture conditions wereoptimized as shown in FIG. 7. Surprisingly, EPCs originate exclusivelyfrom a CD31⁺ subset. Whole BM-MNCs were cultured under the sameconditions and used as an internal control for EPC culture quality.

In addition to the marker study, functional measurements are performedto characterize the endothelial cell phenotype using any one or more ofthe following assays.

Nitric Oxide (NO) Formation

Intracellular nitric oxide (NO) formation is analyzed usingdiamino-fluorescein-2 diacetate (DAF-2 DA, Daiichi, Japan), a membraneNO-specific fluorescence indicator. To compare the intensity offluorescence the image is captured during a fixed exposure time bymanual operation and then get the intensity profile of fluorescence ofeach cell with Image-Pro Plus®.

Vascular Progenitor Cell Culture Assay

CD31⁺ or CD31⁻ cells are resuspended in culture medium, EGM-2 MV, andsubsequently cultured in either EGM-2 MV to induce and maintainendothelial phenotype or EGM-2 supplemented with 10% FBS and plateletderived growth factor (PDGF)-BB stimulation (10 ng/ml, R&D systems) tofacilitate vascular smooth muscle phenotype. After 2 weeks in culture,morphological appearance and immunocytochemical staining are used todefine cellular phenotypes. To detect an endothelial cell phenotype,primary antibodies against von Willebrand factor (vWF) (DAKO), which aredetected with phycoerythrin- (PE-) conjugated goat anti-rat IgG, andantibodies against α-smooth muscle actin (FITC-conjugated, SIGMA) areused to detect vascular smooth muscle cells.

Protective Effects on Cardiomyocytes and Skeletal Myocytes in Co-Culture

For the evaluation of direct protective effects on cell transplantation,CD31⁺ or CD31⁻ cells are co-cultured with cardiomyocytes and satellitecells (muscle precursors) derived from skeletal muscles. Neonatal ratcardiomyocytes are isolated and cultured from syngenic Fisher rats aspreviously described³³ and satellite cells are harvested from adult wildtype C57BL mice⁶⁶. CD31⁺ or CD31⁻ cells are cultured with cardiomyocytesor satellite cells at a ratio of 1:4-1:8 for 7-14 days. Cell culturesare performed under a normoxic conventional condition, ahypoxic-condition, and a hydrogen peroxide (H₂O₂, 100 μM)-stimulatedcondition. Terminal dUTP nick-end labeling (TUNEL) and activated-caspase3 staining are used to quantify anti-apoptotic effects of sorted cellson CMCs and satellite cells⁶⁷. To investigate pro-survival andanti-apoptotic effects of CD31+ or CD31− cells, total Akt,phosphorylated Akt and IGF-1 are measured by Western blot⁶⁸.

Furthermore, lineage-depleted and CD31⁺ or CD31⁻ cells derived from GFPtransgenic mice, which exclude lineage-committed cells such asmonocytes, B and T lymphocytes, are co-cultured to determine thedifferentiation (transdifferentiation) potential of these cells intocardiomyocytes. To confirm the possibility of cell-to-cell fusion inmediating myogenic differentiation of these cells, co-culture withsatellite cells derived from ROSA26 mice will be performed in skeletalmyogenic differentiation media. One day after co-culture, mediasupplement will be changed from fetal bovine serum to horse serum inorder to induce muscle differentiation.

CD31⁺ or CD31⁻ cells derived from GFP expressing transgenic mice(C57BL/6J background) were used in a hindlimb ischemia model and amyocardial infarction model. For a hindlimb ischemia model, both nudemice and syngenic C57BL/6J mice are used. Multifarious analysis isperformed in mice models. Primary endpoints are physiologic andfunctional improvements, such as a reduction in ischemic limb loss, anincrease in hindlimb blood flow, and increases in cardiac systolic anddiastolic function. Secondary endpoints are area of fibrosis andvascular density in histology samples. To track the fates oftransplanted cells in tissues with the use of GFP mice, thedifferentiation potential of transplanted cells is examined usingimmunohistochemical methods. Co-localization of transplanted cells(GFP⁺) with lineage markers (endothelial cell, vascular smooth musclecells, myocytes) identifies transdifferentiated cells.

Example 10 CD31+Cells Possess Greater Efficacy to Repair Ischemic Limbs

CD31+ cells were tested in a mouse hindlimb ischemia model as previouslydescibed⁶⁹. In brief, a ligation was made around the femoral artery andall arterial branches were removed. The consistency of limb ischemia andthe prognosis associated with this model was previously confirmed in ourlaboratory^(7,8). To test the therapeutic effects of cell therapy, CD31⁺or CD31⁻ cells were washed gently with PBS and introduced via tail vein.The dose of injected cells was 1×10⁶ per mouse and the suspension volumewas 200 μL. Whole BM-MNCs and PBS injected mice served as controls. Thechimeric or transdifferentiation potential of injected cells intovasculogenic or myogenic lineages was determined by immunofluorescenthistochemistry. Co-localization of injected cells derived from GFPtransgenic mice with each lineage marker such as von Willebrand factor(for endothelial cells, DAKO), or α-sarcomeric actin (for muscles,Sigma), is indicative of transdifferentiation or chimerism. Untreatedmice consistently lose ischemic hindlimbs following surgery due toimpaired angio-vasculogensis^(69, 70) (FIGS. 9A and 9B). Mice treatedwith CD31⁺ cells were more effective in restoring circulation andrescuing ischemic limbs than CD31⁻ cells (FIGS. 8A, 8B, 9A and 9B).

Doppler perfusion imager (LDPI, Moor instrument, UK), which maps tissueblood flow by the shift in the laser light frequency, was used forserial noninvasive physiological evaluation. After 2 weeks of limbischemia, the CD31⁺ cell group showed increased limb perfusion andcapillary density as well as greater efficacy in the salvage of ischemiclimbs, compared to mice injected with PBS, whole BM cells and CD31⁻cells (n=7, each). Mean values of perfusion were calculated from thestored digital color-coded images. Results of hindlimb blood flow at thetwo week time point was expressed as the ratio of left (ischemic) toright (non-ischemic) to avoid data variations caused by ambient lightand temperature.

Example 11 CD31+ Injection Enhances Function and Healing AfterMyocardial Infarction

Myocardial infarction was induced by ligating the left anteriordescending coronary artery with 8-0 prolene suture. The apex of the leftventrical was observed for evidence of myocardial blanching and akinesiaindicating interruption in coronary flow⁷¹. Immediately after ligation,CD31⁺ (5×10⁵) or CD31⁻ cells with the 50 μl suspended volume wereinjected into the myocardial wall using 27 G needle. All surgicalprocedures were carried out with an operating microscope (Zeiss) at ×5to ×24 magnification. Whole BM-MNCs and PBS injected mice served ascontrols. Mice were assayed at 2 and 4 weeks following myocardialinfarction and cell transplantation. At both time points, systolic anddiastolic functions were improved and fibrosis-scar size was smaller inmice receiving CD31⁺ cells compared to the control groups (n=5, each).

Methods of assaying for efficacy in the treatment of myocardial ischemiaare known in the art. In one example, at post-operative day 14 and 28,echocardiography and pressure transducer measurements are performed.Two-dimensional images and M-mode tracings are recorded from theparasternal short axis view at the level of papillary muscle. FromM-mode tracings, anatomical parameters in diastole and systole areobtained. For hemodynamic studies, the right carotid artery iscannulated with a microtip pressure transducer (Millar 1.4F).

Treatment efficacy will be assessed by measuring fibrosis-scar size.From ischemic limbs, the gastrocnemius muscle is harvested and stainedwith Sirius red, and collagen) volume fraction is measured. In theinfarcted heart, Masson's trichrome staining will be performed on leftventricle samples harvested at 2 and 4 weeks after infarction.Morphometric analysis of fibrosis length and area is performed with acomputerized digital image-analysis system.)

Example 12 CD31⁺ Cells Enhance Angio-Vasculogensis

Isolectin B4 staining (Vector Laboratories) was performed with frozensections of ischemic limbs harvested 2 weeks after ischemic injury.Tissues were counterstained with nuclear DAPI. Lectin staining, whichrepresents capillaries, was used to measure capillary density. Capillarydensity is calculated from at least 5 randomly selected fields. Thenumber of capillaries is converted to the number per square millimeter.As shown in FIGS. 10A and 10B, CD31⁺ cell transplantation group showedincreased capillaries. Also, functionally competent vessels containingvascular smooth muscle layers are identified with an antibody againstα-smooth muscle actin and size (>300 μm²)⁷². All immunostaining isvisualized using conventional inverted fluorescence microscopy and/orlaser scanning confocal microscopy.

To detect S-G2-M phase cells, Ki-67 staining is performed usinganti-Ki-67 antibody (Novocastra Laboratories) with frozen sections oflimbs and left ventricular specimens harvested at 1 and 2 weeks afterischemia. In infarcted heart specimens, the number of positive stainednuclei is measured from both peri-infarct and remote-infarct area. Tomeasure a cumulative fraction of proliferative cell throughout the studyperiod³³, mini-osmotic pump releasing BrdU is implanted and anti-BrdUstaining is performed. To measure apoptosis, a TUNEL assay is performedusing the fluorescein in-situ cell death detection kit (Roche-Molecular)with frozen sections harvested at 1 week after ischemia. In addition toTUNEL, activated, cleaved caspase-3 staining is used to detectapoptosis. To determine the proportion of proliferative or apoptoticnuclei within myocytes (or cardiomyocyte), tissue are counterstainedwith antibodies against α-sarcomeric actin (Sigma) or cardiac Troponin I(Sigma).

Example 13 CD31+Cells Incorporated into Vasculature and ReplenishedVessels in Ischemic Tissues

Donor cells having anti-GFP staining were used to investigate theco-localization of donor cells together with markers for other celltypes, such as endothelial cells, vascular smooth muscle cells,myocytes, and cardiomyocytes. Results shown in FIG. 11 indicated thatCD31⁺ cells likely undergo tissue specific immunophenotypic conversionto replenish vessels in ischemic tissues (FIG. 11).

Vascular lineage differentiation is assayed as follows. Tie-2/LacZtransgenic mice are used as cell donors. Cells of these mice expressβ-gal if endothelial lineage differentiation is determined by Tie-2promoter activation throughout development and in the adult⁷³.

As reported herein, CD31⁺ acts as a comprehensive marker that isexpressed in most if not all of the currently identified BM cellsshowing hemangioblastic activity. These findings suggest that a strategyof CD31⁺ cell transplantation may have significant therapeutic potentialfor repairing ischemic limb and heart.

Example 14 Expression of Angiogenesis Related Genes

The expression of angiongenesis related genes on CD31⁺ cells wasdetermined. BM cells were harvested from mice after euthanasia. CD31⁺and CD31⁻ cells were obtained by magnetic cell sorting (MACS, MiltenyiBiotec, Germany) and subjected to RNA isolation using Trizol. Microarrayanalysis was carried out using a GeneChip Mouse Genome 430 2.0 Array(Affymetrix). 22,691 probe sets were examined and 5,121 probe sets wereexcluded due to weak signal strength compared to the background signal.Among the remaining 17, 658 probesets, 6,132 probesets showedstatistically significant regulation between CD31⁺ vs. CD31⁻. 2,158probesets revealed statistical significance with a fold-change of morethan 2 (1, 290 genes up-regulated and 868 genes down-regulated in CD31⁺cells compared to CD31⁻ cells).

To investigate angiogenic gene expression in CD31⁺ and CD31⁻ cells, weselected 60 angiogenesis-related genes (50 angiogenic genes, 10anti-angiogenic genes) that code secreted proteins or extracellularmatrix proteins (proteins were selected based on literature review andaffymetrix annotation). Among the 60 genes, 43 genes were expressed inCD31⁺ or CD31⁻ cells. Genes that were significantly increased anddecreased in CD31+ cells with more than 1.5-fold were indicated by redand blue letters (Table 1A and B) or triangular dot (FIG. 12),respectively.

TABLE 1A Angiogenic genes UniGeneID Gene Symbol Gene Description Pos/NegMm.1019 Il6 interleukin 6 68.31 Mm.309336 Angpt1 angiopoietin 1 12.92Mm.331089 Pdgfc platelet-derived growth factor, C polypeptide 9.32Mm.268521 Igf1 insulin-like growth factor 1 5.53 Mm.2675 Pdgfa plateletderived growth factor, alpha 3.70 Mm.1402 Vegfc vascular endothelialgrowth factor C 3.09 Mm.1410 Il18 interleukin 18 1.99 Mm.241282 Fgf1fibroblast growth factor 1 1.93 — Ang1 angiogenin, ribonuclease Afamily, member 1 1.88 Mm.795 Csf1 colony stimulating factor 1(macrophage) 1.87 Mm.15607 Vegfb vascular endothelial growth factor B1.65 Mm.144089 Pdgfb platelet derived growth factor, B polypeptide 1.57Mm.137222 Tgfa transforming growth factor alpha 1.39 Mm.267078 Hgfhepatocyte growth factor 1.31 Mm.289681 Hbegf heparin-binding EGF-likegrowth factor 1.26 Mm.377077 Ang2 angiogenin, ribonuclease A family,member 2 1.19 Mm.6813 Bmp4 bone morphogenetic protein 4 1.15 Mm.18213Tgfb2 transforming growth factor, beta 2 1.13 Mm.189536 Angpt4angiopoietin 4 0.95 Mm.390018 Angpt2 angiopoietin 2 0.94 — Tnfsf12 ///tumor necrosis factor (ligand) superfamily, member 12 /// 0.94Tnfsf12-tnfsf13 tumor necrosis factor (ligand) superfamily, member12-member 13 Mm.258415 Nos3 nitric oxide synthase 3, endothelial cell0.91 Mm.32171 Fgf2 fibroblast growth factor 2 0.91 Mm.29564 Mmp2 matrixmetallopeptidase 2 0.90 Mm.57202 Shh sonic hedgehog 0.88 Mm.303231Cxcl12 chemokine (C—X—C motif) ligand 12 0.86 Mm.390122 Pdgfdplatelet-derived growth factor, D polypeptide 0.81 Mm.4956 Fgf4fibroblast growth factor 4 0.79 Mm.173813 Notch4 Notch gene homolog 4(Drosophila) 0.79 Mm.227 Itgav integrin alpha V 0.77 Mm.1810 Ctgfconnective tissue growth factor 0.76 Mm.293761 Pofut1 proteinO-fucosyltransferase 1 0.73 Mm.1238 Csf3 colony stimulating factor 3(granulocyte) 0.68 Mm.87365 Prok2 prokineticin 2 0.68 Mm.4406 Mmp9matrix metallopeptidase 9 0.52 Mm.282184 Vegfa vascular endothelialgrowth factor A 0.40

TABLE 1B Anti-angiogenic genes UniGeneID Gene Symbol Gene DescriptionPos/Neg Mm.332490 Cxcl4 chemokine (C—X—C motif) ligand 4 29.73 Mm.874Il10 interleukin 10 0.98 Mm.26688 Thbs2 thrombospondin 2 0.96 Mm.1421Adamts1 a disintegrin-like and metallopeptidase (reprolysin type) with0.80 thrombospondin type 1 motif, 1 Mm.4159 Thbs1 /// thrombospondin 1/// 0.66 LOC640441 similar to thrombospondin 1 Mm.206505 Timp2 tissueinhibitor of metalloproteinase 2 0.38 — LOC640441 similar tothrombospondin 1 0.31

Example 15 Identity of CD31+ Cells

CD31⁺ cells were characterized as follows.

To characterize the CD31⁺ cells from murine bone marrow, wedouble-stained the BM-derived MNCs with lineage markers and CD31 todetermine what kind of cells compose CD31⁺ cells (FIG. 13). Thepercentage of CD31⁺ cells among CD3e+ (mainly T lymphocytes), B220+(mainly B lymphocytes) and CD14⁺ cells (mainly monocyte/macrophages) ismore than 70%, whereas CD11b+ (mainly monocytes and myeloid) and Gr-1+(mainly granulocytes) cells make up less than 30%. The most dominantcomponent of CD31⁺ cells is B220⁺ cells, which constitute 70% of allCD31+ cells.

Example 16 Blood Flow and Vascularity in the Hindlimb Ischemia Model

A hindlimb ischemia model was used to evaluate blood flow andvascularity.

To induce hindlimb ischemia, ligation was made around the femoral arteryand all arterial branches were removed. To test the therapeutic effectsof cell therapy, CD31⁺ or CD31⁻ cells were washed gently with PBS andinjected intramuscularly. The dose of injected cells was 1×10⁶ per mouseand the suspension volume was 200 μL. Whole BM-MNCs and PBS injectedmice served as controls.

Laser Doppler perfusion imager (LDPI, Moor instrument, UK), which mapstissue blood flow by the shift in the laser light frequency, was usedfor serial noninvasive physiological evaluation. Each mouse was followedby serial recording of surface blood flow immediately after surgery, andat day 3, 7, 14, and 21. Mean values of perfusion were calculated fromthe stored digital color-coded images. The limb blood flow was expressedas the ratio of left (ischemic) to right (non-ischemic) to avoid datavariations caused by ambient light and temperature (FIGS. 14A and 14B).

Isolectin B4 staining (Vector Laboratories) was performed with frozensections of limbs harvested at 2 weeks after ischemic injury (FIGS. 15Aand 15B). Capillary density was calculated from the capillary countsfrom at least 5 randomly selected fields. All immunostaining wasvisualized using conventional inverted fluorescence microscopy and/orlaser scanning confocal microscopy. Tissues were counterstained withnuclear DAPI.

Example 17 Tracking of Engrafted Cells

To determine the phenotypic changes of the transplanted cells, cellsfrom GFP expressing mice were injected into the ischemic hind limb modelof an ischemic hindlimb mouse model. The localization of the injectedcells with anti-GFP staining followed by confocal microscopy wasdetermined. As shown in FIG. 16, CD31⁺ cells successfully incorporatedinto vessels at the location of endothelial cells and pericytes.

Example 18 Transdifferentiation of CD31+ Cells into Endothelial Cells InVitro

To investigate whether CD31⁺ cells can be induced to undergodifferentiation into endothelial cells in vitro, we cultured CD31⁺ cellssupplemented with VEGF-A as outlined in FIG. 17A. CD31⁺ cells underwentdifferentiation into endothelial cells in vitro as demonstrated bychanges in morphology and the expression of endothelial cell specificmarkers (FIGS. 17A and 17B).

Example 19 Evaluation of the Surface Marker Phenotype of Human CD31+ andCD31− Cells

Cells were analyzed with a FACStar flow cytometer. CD31⁺ cells werelabeled with PE- or FITC-conjugated Abs against human CD11b, CD14, CD31,CD45, CD105, CD141, CD144, CD146 or isotype controls. Red lines, controlIg; green line, specific Ab.

Surface Molecule Analysis

To evaluate the surface marker phenotype of CD31⁺ and CD31⁻ cells, cellswere labeled for 20 minutes at manufacture-recommended concentrationswith fluorescent antibodies: monocyte/macrophage markers (anti-CD11b-PE,anti-CD14-PE), hematopoietic lineage maker (anti-CD45-FITC) andendothelial cell markers (anti-CD141-PE, anti-CD105-PE, anti-CD144-FITCand anti-CD146-PE). As a negative control, fluorescent isotype matchedantibodies were used. Cells were rinsed, paraformaldehyde-fixed, andanalysis was performed by FACS-Calibur Instrument (Becton-Dickins).

hCD31⁺ (human CD31+) cells were isolated using a magnetic beadseparation technique. To define the phenotype of hCD31⁺ cells,Fluorescence-activated cell sorting (FACS; BD bioscience) analysis wasperformed. FACS analysis demonstrated that greater than 40% of the cellsinclude monocyte/macrophage markers (CD11b, CD14), and endothelial cellmarkers (CD141, CD105, and CD144), but not CD146 (FIG. 18). In addition,more than 98% of hCD31⁺ cells are positive for CD45, suggesting thathCD31⁺ cells are not circulating endothelial cells (FIG. 18). Incontrast, hCD31⁻ cells expressed low levels of CD105 and CD144. HSCmarkers such as CD34, CD133, KDR, Tie-2 were not present on eitherhCD31⁺ or hCD31⁻ cells.

Example 20 Real Time PCR-Evaluation of Endothelial and InflammatoryGenes in Human CD31+ and CD31− Cells

Real time PCR analysis of specific endothelial and inflammatory genes inCD31+ and CD31 cells was performed.

Quantitative Real-Time PCR Assay

Total RNA was isolated from peripheral blood CD31⁺ cells and CD31⁻ cellsby the use of RNA-stat (Iso-Tex Diagnostics) according to themanufacture's instructions. Subsequently, extracted RNA wasreverse-transcribed by use of Taqman Reverse Transcription Reagents(Applied Biosystems) for cDNA synthesis. For real-time reversetranscription-polymerase chain reaction (RT-PCR), human-specific primersand probes, respectively (see supplemental table 1) were used.Quantitative assessment of RNA levels were performed by use of an ABIPRISM 7000 Sequence Detection System. The relative expression value oftarget, normalized to the endogenous control GAPDH (house-keeping) geneand relative to a calibrator, is expressed as the fomular RelExp=2^(−ΔCT) (fold difference), where ΔCt=(Ct of target genes)−(Ct ofendogenous control gene, GAPDH) in experimental samples. The number ofPCR cycles was measured using Lightcycler 3.5 software.

To investigate the expression of multiple angiogenic and inflammatorygenes from the hCD31⁺ population, we measured mRNA levels usingreal-time PCR. The expression levels of Angiopoietin-1, HGF, VE-CadherinVEGF-A, interferon-γ and TNF-α significantly increased in the hCD31⁺group compared to the hCD31⁻ group.

CD31+ cells are highly enriched with mRNA for endothelial genes, buthave a decreased level of mRNA for inflammatory genes, as compared withCD31− cells. Equivalent amounts of RNA were used for gene comparisons.All of the assays were performed in triplicate. (the CD31+ cells of 3donors were pooled). Data are Mean±SEM. *P<0.05, **P<0.01 vs. CD31−.Results are shown in FIG. 19.

Example 21 Characteristics of EPC and Functional Analysis of HumanPeripheral Blood CD31 Derived Cells

EPCs were characterized and a functional analysis of peripheral bloodCD31 derived cells was performed (FIG. 20). The number of adherent (AD)cells and colonies in CD31+ and CD31− cells was compared. Phase-contrastimages show significantly greater numbers of adherent cells and coloniesin CD31⁺ cells derived from equivalent numbers of cells examined at 7days of culture. (n=5, **P<0.01). An EPC culture assay was performed.FIG. 20C shows CD31⁺ cells were cultured for 4 days then identified asadherent cells double positive for DiI-acLDL uptake (red) and UEA-1lectin binding (green). FIG. 20D shows that the CD31⁺ group demonstrateda significantly greater number of EPC cells compared with CD31⁻ group(n=4, **P<0.01). FIG. 20E shows results of a colony-forming EPC assaythat was performed. Colony-forming EPCs were evaluated by performing anendothelial cell differentiation culture with CD31+ or CD31− cells. Theupper and lower panel shows a typical colony of EPCs double positive forDiI-acLDL uptake (red) and isolectin B4-FITC binding (green), appearingyellow on merged images. In FIG. 20F, the right panel shows the countsof double-positive colonies grown from CD31+ and CD31− group,respectively (n=3; **P<0.01). FIG. 20G in vitro differentiation of CD31+cells into endothelial cells was examined. Immunofluorescent imagingdemonstrated that CD31⁺ cells expressed EC-specific proteins, such asvWF, VE-cadherin, KDR and CD31. Nuclei were counterstained with DAPI inblue. FIG. 20H shows the incorporation of CD31⁺ cells into the HUVECnetwork was determined. DiI-labeled attaching CD31⁺ cells wereincorporated into the non-labeled HUVEC network on basement matrix gelat 24 hours of coculture.

Colony Forming Unit-Fibroblast (CFU-F) Assay

To evaluate the potential of EPC colony formation, 5×10⁶ MACS sortedCD31+ or CD31− cells were cultured in a 6-well plate for 4 days. Cellswere incubated with DiI-acLDL (1:500, Biomedical Technologies) for 1 h,then washed with PBS, fixed in 1% paraformaldehyde, and stained withUEA-1 lectin (1:200, sigma). DiI-acLDL uptake and UEA-1 lectin bindingdouble positive cell colonies were counted. CD31⁺ cells and CD31⁻ cellswere cultured in 2-well glass slides at 5×10⁶ cells per well and mediumwas changed between 7 and 10 days. Aggregates of 30 cells or more werescored as CFU-F.

In Vitro Endothelial Cells Differentiation

To induce endothelial lineage differentiation, total hCD31⁺ cells werecultivated in plastic dishes in DMEM with low (1 g) glucose containing15% of FBS for 2 weeks, supplemented with 25 ng/ml HGF, EGF, FGF, VEGF,IGF-1 and ascorbic acid.

Endothelial Network Formation on Matrix Gel Culture

After 7 day of culture, peripheral blood CD31⁺ adherent cells wereobtained from culture plates. CD31⁺ cells were then labeled with DiI andcocultured with unlabeled human umbilical vein endothelial cells(HUVECs) on basement membrane matrix gel (Matrigel™, Becton, Dickinson)at a 1:10 ratio. After 24 hours of incubation, endothelial networkformation and incorporation of red fluorescent-labeled CD31⁺ cells intoendothelial networks were examined and representative fields werephotographed under fluorescence microscopy.

When human peripheral blood derived hCD31⁺ cells (n=7) were cultured onplastic dishes, a number of adherent cell and colonies appeared after 4day of culture (FIGS. 20A-B). Much greater numbers of adherent cells andcolonies developed from hCD31⁺ (n=7) than from the same amount of hCD31⁻cells (n=7; P<0.01) (FIGS. 20A-B). We first hypothesized that themajority of isolated hCD31⁺ cells contain primitive EPCs. To identifywhether hCD31⁺ cells contain primitive EPCs, we used equal numbers ofhCD31⁺ and hCD31⁻ cells from human peripheral blood, then performed EPCand EPC colony culture assays. The EPCs and EPC colonies were identifiedby double staining for DiI-acLDL uptake and UEA1-FITC binding (FIGS.20C-F). Significantly greater number of EPC and EPC colonies weregenerated from the hCD31⁺ cell population compared with hCD31⁻ cellspopulation (n=7; P<0.01).

Human peripheral blood hCD31⁺ cells were isolated (purity 95%-98%) andcultivated under endothelial conditions. To induce endothelial lineagedifferentiation, total hCD31⁺ cells were cultivated in plastic dishes inDMEM with low (1 g) glucose containing 15% of FBS, supplemented with 25ng/ml HGF, EGF, FGF, VEGF, IGF-1 and ascorbic acid. To determine ifendothelial cell differentiation had occurred, the expression of aselected set of markers was characterized by flow cytometry after 10-15days serial culture. Immunofluorescencent cytochemistry assays revealedthat hCD31⁺ cells exhibited the endothelial cell markers such asVe-Cadherin, KDR, vWF, CD31 fourteen days after culture. (FIG. 20G).

The angiovasculogenic function of hCD31⁺ cells cultivated underendothelial conditions in vitro was examined. To investigate whetherhCD31⁺ cells participated in endothelial network formation, matrigeltube formation assays were performed. hCD31⁺ cells were collected at day14 of culture, labeled with the red fluorescent dye CM-Dil (Dil), andco-cultured with HUVECs on basement membrane matrix gel. At 24 hours ofco-culture, hCD31⁺ cells incorporated with HUVEC networks (FIG. 20H).

Example 22 In Vitro Differentiation of Human CD31+Cells intoVascular-Like Tube After 4 Weeks in EPC Culture Condition

The differentiation of CD31+ cells into vascular tubes was observed.

In Vitro Endothelial Cells Differentiation

To induce endothelial lineage differentiation, total hCD31⁺ cells werecultivated in plastic dishes in DMEM with low (1 g) glucose containing15% of FBS for 2 weeks, supplemented with 25 ng/ml HGF, EGF, FGF, VEGF,IGF-1 and ascorbic acid.

Cells were incubated with DiI-acLDL (1:500, Biomedical Technologies) for1 h, then washed with PBS, fixed in 1% paraformaldehyde, and stainedwith UEA-1 lectin (1:200, sigma) and DAPI (1:5000).

CD31+ cells formed vascular-like tubes after 4 weeks in EPC culturecondition (FIG. 21A-F). CD31+ cells changed sequentially intovascular-like tubes (FIG. 21, A-B). CD31⁺ cells are capable of formingcapillaries in vitro.

The cells of the vascular-like tube were identified by double stainingfor DiI-acLDL uptake and UEA1-FITC binding (FIG. 21C-E).Immunohistochemistry of CD31⁺ cell-derived vascular-like tubes showsthat they expressed UEA-1 lectin and incorporated Dil-Ac-LDL like tubesgrown from endothelial cell lines. Nuclei counterstained with DAPI inblue.

Example 23 Real Time PCR of Specific Endothelial and Inflammatory Genesin Endothelial Cell Differentiated CD31⁺ Cells

Real time PCR of specific endothelial and inflammatory genes wasperformed in endothelial cell differentiated CD31⁺ cells.

CD31+ cells were cultured in EPC culture medium for 4 weeks.Non-adherent cells were discarded and only adherent cells were analyzed.Equivalent amounts of RNA were used for all gene comparisons. All of theassays were performed in triplicate.

Total CD31⁺ cells were cultivated in plastic dishes in DMEM with low (1g) glucose containing 15% FBS for 4 weeks, supplemented with 25 ng/mlHGF, EGF, FGF, VEGF, IGF-1 and ascorbic acid. All samples were collectedevery week. RNA was isolated with RNA-stat (Iso-Tex Diagnostics)according to the manufacture's instructions. Subsequently, extracted RNAwas reverse-transcribed by use of Taqman Reverse Transcription Reagents(Applied Biosystems) for cDNA synthesis. For real-time reversetranscription-polymerase chain reaction (RT-PCR), we used human-specificprimers and probes, respectively (see supplemental table 1).Quantitative assessment of RNA levels was performed by use of an ABIPRISM 7000 Sequence Detection System. The relative expression value oftarget, normalized to the endogenous control GAPDH (house-keeping) geneand relative to a calibrator, is expressed as the fomular RelExp=2^(−ΔACT) (fold difference), where ΔCt=(Ct of target genes)−(Ct ofendogenous control gene, GAPDH) in experimental samples. The number ofPCR cycles was measured using Lightcycler 3.5 software.

Human peripheral blood hCD31⁺ cells were isolated (purity 95%-98%) andcultivated under endothelial conditions. To induce endothelial lineagedifferentiation, total hCD31⁺ cells were cultivated in plastic dishes inDMEM with low (1 g) glucose containing 15% of FBS, supplemented with 25ng/ml HGF, EGF, FGF, VEGF, IGF-1 and ascorbic acid. Endothelial celldifferentiation was analyzed by characterizing the expression of aselected set of markers by real-time PCR after 4 weeks of serial culture(FIG. 22). Endothelial-like cells derived from CD31 cells becomepositive for SDF-1α, IGF-1 and negative for TNF-α, IFN-γ duringdifferentiation.

Example 24 In Vivo Vasculogenesis of Human CD31 Cells in Ischemic Limbof Nude Mouse

The in vivo vasculogenesis of CD31+ cells in an ischemic limb of a nudemouse was determined.

All experimental protocols were approved by Caritas St. Elizabeth'sInstitutional Animal Care and Use Committee (IACUC). Female athymic nudemice (Charles River Laboratories), 6 to 9 weeks old and 18 to 22 g inweight were used. To induce a hindlimb ischema model, mouse wereanesthetized with 120 mg/kg intraperitoneal pentobarbital for operativeresection of one femoral artery. Surgery to create hindlimb ischemia wascompleted by resecting the right femoral arteries. Mice weretransplanted with DiI-labeled CD31⁺, CD31⁻ cells in EBM medium or PBSintramuscularly into the ischemic hindlimb area after surgery (n=9 foreach implantation). To measure serial blood flow in hindlimb for the 4weeks after operation, Laser Doppler perfusion image analyzer (MoorInstrument, Wilmington, Del.) was used. After 28 days mice in each groupwere sacrificed.

Therapeutic Effects of Human CD31+ Cells

To investigate the therapeutic potential of CD31⁺ cells hindlimbischemia was surgically induced in athymic nude mice. hCD31⁺ (humanCD31⁺) cells, hCD31⁻ (human CD31⁻) cells and PBS were injected into theischemic hindlimbs (n=9, each). The results are presented in FIGS.23A-23F.

The hCD31⁺ treated group showed higher limb salvage rate than othergroups [total salvage/tip necrosis/amputation; hCD31⁺ cells 5/4/0,hCD31⁻ cells 2/5/2, PBS control 0/5/4] (n=9 each group).

LDPI analysis revealed that a greater degree of blood perfusion wasobserved in the ischemic limb of hCD31⁺ cells injected mice (59%increase at day 21, P 0.001) compared with hCD31− cell or PBStransplanted control mice on days 7 and 14. Capillary densities werealso measured in tissue sections collected at day 14 from the lowerabductor of ischemic hindlimb. The overall capillary density of thehCD31⁺ treated group was also significantly higher than hCD31⁺ and PBStreated groups (FIG. 23). These data suggest implantation of CD31⁺ cellsnot only prevents adverse vascular remodeling, but showsangio-vasculogenic potential in vivo.

Example 25 Expression of Angiogenic Factors Following CD31+Transplantation

The expression of angiogenic factors following CD31+ transplantation wasdetermined as follows.

Quantitative Real-Time PCR Assay

Total RNA was isolated from each of CD31⁺, CD31⁻ and PBS injectedhind-limb tissues after 7 days by the use of RNA-stat (Iso-TexDiagnostics) according to the manufacture's instructions. Subsequently,extracted RNA was reverse-transcribed with Taqman Reverse TranscriptionReagents (Applied Biosystems) for cDNA synthesis. For real-time reversetranscription-polymerase chain reaction (RT-PCR), human-specific primersand probes, respectively (see supplemental table 1) were used.Quantitative assessment of RNA levels were performed by use of an ABIPRISM 7000 Sequence Detection System. The relative expression value oftarget, normalized to the endogenous control GAPDH (house-keeping) geneand relative to a calibrator, is expressed as the fomular RelExp=2^(−ΔCT) (fold difference), where ΔCt=(Ct of target genes)−(Ct ofendogenous control gene, GAPDH) in experimental samples. The number ofPCR cycles was measured using Lightcycler 3.5 software.

Multiple Angiogenic Factors are Upregulated After CD31⁺ CellsTransplantation

To determine the level of expression of various cytokines following celltransplantation into the ischemic hindlimb of athymic nude mice, micewere sacrificed and hindlimb tissues were collected. The expressionlevels of VEGF-A, FGF-2 (bFGF), Angiopoitin-1, PDGF and e-NOS weresignificantly increased in the CD31⁺ cell injected group compared toCD31⁻ cells and PBS injected group (FIG. 24). Overall, the expression ofmultiple angiogenic, chemoattractant cytokines was greater in thehindlimb of CD31⁺ injected group compared to CD31⁻ and PBS injectedgroups (FIG. 24). However, the expressions of inflammatory genes such asIL-1,6,10 and IFN-γ revealed no difference among these three groups(FIG. 24).

Example 26 Transdifferentiation of Human CD31+ Cells into EndothelialCells

The transdifferentiation of CD31+ cells into endothelial cells wasanalyzed as follows.

Histological Analysis

Mice were killed 2 weeks after cells transplantation. For capillarydensity measurement, four frozen sections of ischemic tissue from theadductor and semimembranous muscles from each group were stained withprimary biotinlated isolectin B4 and secondary strepta-avidin Alexafluor488 (Invitrogen). Five fields from four tissue sections were randomlyselected, and the number of capillaries was counted in each field.Pictures were photographed using a fluorescent inverted microscopy or aconfocal microscopy. Ten mice were used to define whether administeredCD31⁺ or CD31⁻ cells differentiated into endothelial cells.

Transdifferentiation of hCD31⁺ Cells into Endothelial Cells

To define the transdifferentiation potential of hCD31⁺ cells in HLImice, 1×10⁶ Dil-labeled hCD31⁺ cells were transplanted intramuscularlyinto the ischemic hindlimb of nude mice. Histologic analysisdemonstrated that a fraction of injected Dil-labeled hCD31⁺ cellsexhibit endothelial phenotypes during the follow-up period of 2-8 weeks.

FIGS. 25A and 25B present the results of immunofluorescence analyis ofreplicate samples of CD31⁺ cells. Immunofluorescence staining imagesshow that human peripheral blood CD31⁺ cells were transdifferentiatedinto endothelial cells lineage. Tissue sections were from hind limbharvested at 2 weeks from animals with hindlimb ischemia followed by theintramuscular injection of Dil-labeled CD31 cells (red). Sections werestained for ILB-4 (Green) which is endothelial cell marker. For nucleidetection, DAPI (blue) was used as a countertain. Bar: 50 um

Example 27 Evaluation of Apoptosis in CD31+ and CD31− Cells

Quantitative analysis of TUNEL-positive cells was performed as follows.

To evaluate apoptosis, TdT-mediated dUTP nick-end labeling (TUNEL)reaction was performed by using the fluorescein in situ cell deathdetection kit (Roche-Molecular).

hCD31⁺ Cells Transplantation Decreases Apotosis

To determine if hCD31⁺ cell transplantation decreases apoptosis, TUNELassays with tissue sections harvested from CD31+, CD31− or PBStransplanted hindlimbs at day 7 following transplantation were performed(FIGS. 26A and 26B). The number of TUNEL positive nuclei in the hindlimbischemia was almost 3 times lower in the CD31⁺ group than in the PBSgroup.

Example 29 Real Time PCR Analysis of Human Bone Marrow CD31+ Cells

The expression of multiple angiogenic and inflammatory genes from thebone marrow derived hCD31⁺ population was investigated as follows.

Total RNA was isolated from human bone marrow derived CD31⁺ cells andCD31⁻ cells by the use of RNA-stat (Iso-Tex Diagnostics) according tothe manufacture's instructions. Subsequently, extracted RNA wasreverse-transcribed by use of Taqman Reverse Transcription Reagents(Applied Biosystems) for cDNA synthesis. For real-time reversetranscription-polymerase chain reaction (RT-PCR), human-specific primersand probes, respectively (see supplemental table 1) were used.Quantitative assessment of RNA levels were performed by use of an ABIPRISM 7000 Sequence Detection System. The relative expression value of atarget, normalized to the endogenous control GAPDH (house-keeping) geneand relative to a calibrator, is expressed as the fomular RelExp=2^(−ΔCT) (fold difference), where ΔCt=(Ct of target genes)−(Ct ofendogenous control gene, GAPDH) in experimental samples. The number ofPCR cycles was measured using Lightcycler 3.5 software.

To investigate the expression of multiple angiogenic and inflammatorygenes from the hCD31⁺ population, mRNA levels were measured usingreal-time PCR. The expression levels of IGF-1, VEGF-A and CD31 weresignificantly higher in the CD31+ group and the expression ofinterferon-γ and TNF-α was significantly decreased in the hCD31⁺ groupcompared to the hCD31⁻ group (FIG. 27).

Example 30 Characterization of BM-Derived Lin− Cells

To determine if BM-derived CD31 expressing cells include multipotentprogenitor and stem cells, CD31⁺ and Lineage-depleted CD31⁺ (Lin⁻CD31⁺)cells were isolated from BM of adult C57BL/6J mice and analyzed for theexpression of stem cells markers. Lin⁻ CD31⁺ cells from mouse BM expressvery high levels of stem cell markers, including c-kit, Scal-1 andFlk-1. CD31 expressing subsets, including CD31⁺, Lin⁻CD31⁺ andLin⁻CD31⁺Sca-1⁺ Lin−CD31+ cells from mouse BM are also highly enrichedfor hemotopoietic clonogenic progenitor cells and can be efficientlyexpanded when cultured with hematopoietic growth factors. In an in vivocongenic competitive bone marrow transplantation model, lin⁻CD31⁺ cellsrevealed greater potential to repopulate lethally irradiated bonemarrow.

BM-Lin⁻ and −Lin⁻c-kit⁺Sca-1⁺ Cells Express Very High Levels of CD31.

BM cells were obtained from BM of C57BL/6J mice and were partiallylineage-depleted by staining BM cells with mouse biotinilated lineagemAbs followed by anti-biotin microbeads. Partially depleted Lin⁻ cellswere then incubated with mouse mAbs specific for Scal-1, c-kit and CD31and analyzed by flow cytometry (FIG. 28). These data demonstrate thatboth Lin⁻ and Lin⁻Sca-1⁺ c-kit⁺ cells express high levels of CD31.

BM-Derived Lin⁻CD31⁺ Cells Express Multiple Stem Cell Markers, Such asc-kit, Sca-1 and Flk-1.

To investigate CD31 as a stem cell marker, BM cells were partiallylineage-depleted by staining of BM cells with mouse biotinilated lineagemAbs followed by anti-biotin microbeads. Partially depleted Lin⁻ cellswere incubated with mouse mAbs, including APC-lineage cocktail,PE-Scal-1, PE-c-kit, PE-Flk-1, FITC-CD31 and CD45, and then analyzed byflow Cytometry (FIG. 29). These data demonstrated that Lin⁻CD31⁺ cellsexpress high levels of c-kit, and also Scal-1 and Flk-1, indicating thatLin⁻CD31⁺ cell likely have multipotent stem cell potential.

Example 31 Hematopoietic Clonogenic Potential of CD31 Expressing Cells

Mouse BM-derived CD31 expressing subsets are highly enriched forhematopoietic progenitor cells and possess greater hematopoieticclonogenic ability when compared with CD31⁻ negative populations ofcells. To examine the hematopoetic clonogenic potential of CD31expressing cells, CD31⁺ and Lin⁻CD31⁺ cells were isolated from MB cellsof C57BL/6J mice using magnetic beads (Miltenyi Biotech). The purity ofboth CD31⁺ and Lin⁻CD31⁺ cells is >95%. 1×10⁴ of CD31⁺ or 1×10³ ofLin⁻CD31⁺ cells were plated in 35 mm Petri dishes with completeMethoCult Media (Stem Cell Technologies), containing rmCSF (50 ng/ml),rmIL-3 (10 ng/ml), rhIL-6 (10 ng/ml) and rhEPO (3 U/ml), and incubatedfor 5 to 7 days at 37° C., 5% CO₂ and >95% humidity. Colonies wereidentified and counted using an inverted microscope (FIG. 30). Thesedata show that CD31⁺ and Lin⁻CD31⁺ cells give rise to significantly morecolonies, including CFU-E, CFU-GM and CFU-GEMM, when compared to CD31nonexpressing cells (FIG. 30).

Example 32 Expansion of CD31⁺ Cells

CD31 expressing cells, including CD31⁺, Lin⁻CD31⁺ and Lin⁻CD31⁺ Sca-1⁺cells, can be efficiently expanded when cultured with hematopoieticgrowth factors and have multipotent hematopoietic differentiationpotential with Lin⁻CD31⁺ and Lin⁻CD31⁺ Sca-1⁺ cells.

To examine the hematopoietic proliferation potential of CD31 expressingcells, CD31⁺ and Lin⁻CD31⁺ were sorted by MoFlo and cultured with theIMDM medium, containing rmTPO (20 ng/ml), rmSCF (20 ng/ml) and rmFlt3-L(20 ng/ml). At different time points cells were counted and analyzed byFlow cytometry (FIG. 31). These results showed that CD31⁺, Lin⁻CD31⁺grow rapidly under the culture condition with hematopoietic growthfactors. However, CD31⁻, Lin⁻CD31⁻ cells did not proliferate and died in3 to 5 days (FIG. 31 a,b).

To further examine whether Lin⁻CD31⁺ Sca-1⁺ cells are enriched forhematopoietic stem cells, Lin⁻CD31⁺ Sca-1⁺ cells were sorted by MoFloand cultured as described above. Flow cytometry analysis showed thatLin⁻CD31⁺ Sca-1⁺ cells grow rapidly. However, Lin⁻CD31⁺Sca-1⁻ cells didnot grow (FIG. 31 f). In addition, multiple lineage differentiation werealso demonstrated when Lin⁻CD31⁺ and Lin⁻CD31⁺ Sca-1⁺ cells werecultured (FIG. 31 c,d, g).

Example 33 Lin⁻CD31⁺ Cells Have Greater Potential to Repopulate BM ofC57BL/6 Mice In Vivo

To further text the ability of Lin⁻CD31⁺ cells to repopulate BM in vivo,Lin⁻CD31⁺ were sorted from the BM of C57BL/6J-CD45.2 mice by MoFlo. 0.5to 5×10⁴ of Lin⁻CD31⁺ cells together with 1×10⁵ fresh BM cells fromCD45.1 C57BL/6J mice were transplanted into lethally irradiated C57BL/6JCD45.1 mice intravenously. Mice injected with either 5×10⁴ of Lin⁻CD31⁺cells together with 1×10⁵ of fresh BM cells or 1×10⁵ BM cells aloneserved as control groups.

To determine the contribution of the transplanted Lin⁻CD31⁺ or Lin⁻CD31⁻cells CD45.2 BM cells, to the reconstitution of total BM chimeras, ormyeloid and lymphoid compartments of transplantation chimeras, cellswere obtained from peripheral blood 5 weeks after transplantation and,after red blood lysis, stained with FITC-conjugated antibody to CD45.2and PE-conjugated antibodies to CD45.1, Mac-1, GR-1, B-220, TER-119 orCD 90.2, and then analyzed by Flow Cytometry (FIG. 32). These datashowed that mice injected with Lin⁻CD31⁺ cells obtained higher levels ofreconstitution of total BM chimeras than mice injected Lin⁻CD31⁻ cells,64% and 44% respectively (P<0.05) (FIG. 32A) and the reconstitutions aremultiple-lineage, including myeloid and lymphoid lineages. FIG. 32Bshowed a representative dot blot analysis of transplanted mice forreconstitution of total BM chimeras. Our experiments indicate thatLin⁻CD31⁺ cells have greater multipotent hematopoietic repopulatingpotential, as compared to Lin−CD31− cells.

Results described herein were obtained using the following methods andmaterials.

Methods and Materials

Mice ranging from 6-8 weeks of age were used. Bone marrow (BM) cellswere isolated from male mice of either GFP-expressing C57BL/6Jbackground or wild type. Isolated cells were used as donors in studiesof transplantation or gene expression/cell culture/flow cytometricanalysis. Female mice of either C57/BL6J wild type or athymic nude micereceived cell transplantation after BM ablating radiation, myocardialinfarction or limb ischemia.

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Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A method of repairing or regenerating a tissue in a subject in needthereof, the method comprising contacting the tissue with a CD31⁺ cell,thereby repairing or regenerating the tissue.
 2. A method for increasingangiogenesis in a subject in need thereof, the method comprisingcontacting the tissue with a CD31⁺ cell, thereby increasingangiogenesis.
 3. The method of claim 1 or 2, wherein the tissue is amuscle tissue, skeletal muscle tissue, cardiac tissue, neural tissue,liver tissue, pancreatic tissue, bone tissue, cartilage, renal tissue,eye tissue, skin tissue or a tissue characterized by excess cell death.4. The method of claim 1 or 2, wherein the subject has or has apropensity to develop a disease selected from the group consisting ofmyocardial infarction, congestive heart failure, peripheral vascularobstructive disease, ischemia, limb ischemia, stroke, transientischemia, reperfusion injury, peripheral neuropathy, diabeticneuropathy, toxic neuropathy, diabetic dementia, or autonomicneuropathy, spinal cord injury, leukemia, lymphoma, myelodysplasticsyndrome, pancytopenia, anemia, thrombocytopenia, leukopenia, liverfailure, renal failure, diabetes, rheumatoid arthritis, osteoarthritis,skin wound, diabetic foot or ulcer, gangrene, diabetic wound andosteoporosis.
 5. A method for ameliorating ischemia related tissuedamage in a subject in need thereof, the method comprising: (a)administering to the subject a CD31⁺ cell; and (b) increasingangiogenesis in a tissue of the subject, thereby ameliorating ischemiarelated tissue damage in the subject.
 6. A method for amelioratingischemia related tissue damage in a subject in need thereof, the methodcomprising: (a) administering to the subject a CD31⁺ cell; and (b)increasing secretion of a paracrine factor or cytokine in a tissue ofthe subject, thereby ameliorating ischemia related tissue damage in thesubject.
 7. The method of claim 5 or 6, wherein the ischemia relatedtissue damage is associated with heart failure, myocardial infarction,other ischemic heart diseases, limb ischemia, stroke, transientischemia, or reperfusion injury.
 8. A method for ameliorating aneuropathy in a subject in need thereof, the method comprising: (a)administering to the subject a CD31⁺ cell; and (b) increasingangiogenesis in a neural tissue of the subject, thereby ameliorating aneuropathy in the subject.
 9. A method for ameliorating a neuropathy ina subject in need thereof, the method comprising: (a) administering tothe subject a CD31⁺ cell; and (b) increasing secretion of a paracrinefactor or cytokine in a neural tissue of the subject, therebyameliorating a neuropathy in the subject.
 10. A method for amelioratingheart failure in a subject in need thereof, the method comprising: (a)administering to a cardiac tissue a CD31⁺ cell; and (b) increasingangiogenesis in the cardiac tissue, thereby ameliorating heart failurein the subject.
 11. A method for ameliorating heart failure in a subjectin need thereof, the method comprising: (a) administering to a cardiactissue a CD31⁺ cell; and (b) increasing myogenesis in the cardiactissue, thereby ameliorating heart failure in the subject.
 12. A methodfor ameliorating heart failure in a subject in need thereof, the methodcomprising: (a) administering to a cardiac tissue a CD31⁺ cell; and (b)increasing secretion of a paracrine factor or cytokine in the cardiactissue, thereby ameliorating heart failure in the subject.
 13. A methodfor increasing wound healing in a tissue of subject in need thereof, themethod comprising: administering to said tissue a CD31⁺ cell therebyincreasing wound healing.
 14. A method for increasing wound healing in atissue of a subject in need thereof, the method comprising: (a)administering to said tissue a CD31⁺ cell; and (b) increasingangiogenesis thereby increasing wound healing.
 15. A method forincreasing wound healing in a tissue of a subject in need thereof, themethod comprising: (a) administering to said tissue a CD31⁺ cell; and(b) increasing secretion of a paracrine factor or cytokine in saidtissue, thereby increasing wound healing in the subject.
 16. A methodfor increasing wound healing in a tissue of a subject in need thereof,the method comprising: (a) administering to said tissue a CD31⁺ cell;and (b) engrafting the CD31+ cell into the tissue, thereby increasingwound healing.
 17. A method for treating a hematologic disease in asubject in need thereof, the method comprising administering to saidtissue a CD31+Lin− cell thereby treating said hematologic disease. 18.The method of claim 17, wherein said hematologic disease is selectedfrom the group consisting of: leukemia, lymphoma, myelodysplasticsyndrome, pancytopenia, anemia, thrombocytopenia, leucopenia.
 19. Amethod for ameliorating liver or renal failure in a subject in needthereof, the method comprising: (a) administering to a liver or renaltissue a CD31⁺ cell; and (b) engrafting the CD31⁺ cell into the liver orrenal tissue, thereby ameliorating liver or renal failure in thesubject.
 20. The method of any one of claims 1 to 19, wherein the cellis integrated into the tissue.
 21. The method of any one of claims 1 to19, wherein the CD31⁺ cell is isolated and expanded in vitro to obtain acell population enriched in bone marrow-derived stem or progenitor cellsprior to being administered to the host subject.
 22. The method of anyone of claims 1 to 19, wherein the CD31+ cell is lineage depleted. 23.The method of any one of claims 1 to 19, wherein the cell is geneticallymodified.
 24. The method of any one of claims 1 to 19, wherein the cellis an endothelial progenitor cell (EPC).
 25. The method of any one ofclaims 1 to 19, wherein the cell is isolated from bone marrow,peripheral blood or umbilical cord blood of a donor subject.
 26. Themethod of any one of claims 1-19, wherein said donor is a mammal. 27.The method of any one of claims 1-19, wherein said donor is a human. 28.The method of any one of claims 1 to 9, wherein the donor and thesubject receiving the cell are the same individual.
 29. The method ofany one of claims 1 to 9, wherein the cell is a human multipotent stemcell expressing or having altered levels of a marker selected from thegroup consisting of: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4,GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHCclass II receptor, as compared to a CD31-cell.
 30. The method of claim16, wherein the cell expresses reduced levels of at least two, three,four, or more markers.
 31. The method of any one of claims 1 to 19,wherein the cell expresses at least one of Sca-1 or c-kit.
 32. Themethod of any one of claims 1 to 19, wherein the cell does not expressLin.
 33. The method of any one of claims 1 to 19, further comprisingadministering to the host subject a therapeutic polypeptide or a nucleicacid encoding a therapeutic polypeptide.
 34. The method of any one ofclaims 1 to 19, wherein the cell is locally or systemicallyadministered.
 35. A method for identifying a multipotent stem cell, themethod comprising: identifying a cell that expresses CD31⁺.
 36. A methodfor identifying a multipotent stem cell, the method comprising:identifying a cell that expresses CD31⁺ and does not express Lin. 37.The method of claim 35 or 36, wherein said identified cell expresses atleast one of Sca1 and c-kit.
 38. The method of claim 35 or 36, whereinthe identification step involves an immunoassay.
 39. A method ofisolating a multipotent stem cell the method comprising: a) isolating aCD31+ cell; and b) selecting said CD31+ cell.
 40. A method of isolatinga multipotent stem cell the method comprising: a) isolating a CD31⁺lin⁻cell; and b) selecting said CD31⁺lin⁻ cell.
 41. The method of claim 39or 40 further comprising a step of culturing said cell of step (a) priorto said selection step.
 42. A packaged pharmaceutical comprising atherapeutically effective amount of a CD31⁺ cell, and instructions foruse in treating a subject, wherein the subject has or has a propensityto develop a disease selected from the group consisting of myocardialinfarction, congestive heart failure, peripheral vascular obstructivedisease, ischemia, limb ischemia, stroke, transient ischemia,reperfusion injury, peripheral neuropathy, diabetic neuropathy, toxicneuropathy, diabetic dementia, or autonomic neuropathy, spinal cordinjury, leukemia, lymphoma, myelodysplastic syndrome, pancytopenia,anemia, thrombocytopenia, leukopenia, liver failure, renal failure,diabetes, rheumatoid arthritis, osteoarthritis, skin wound, diabeticfoot or ulcer, gangrene, diabetic wound and osteoporosis.
 43. Thepackaged pharmaceutical of claim 42, further comprising a therapeuticpolypeptide.
 44. The packaged pharmaceutical of claim 42 or 43, whereinthe cell is genetically modified.
 45. The packaged pharmaceutical ofclaim 42 or 43 wherein said cell is Lin−.
 46. The packagedpharmaceutical of claim 42 or 43 wherein said cell expresses at leastone of Sca1 or c-kit.
 47. A method for identifying an agent useful forenhancing the transdifferentiation of a CD31⁺ cell, the methodcomprising (a) contacting a CD31⁺ cell with an agent; and (b) measuringan increase in the expression of a protein not expressed in an untreatedCD31⁺ control cell, wherein an increase in protein expression in saidCD31+ cell of step (a) as compared to said CD31+ control cell identifiesthe agent as useful for transdifferentiating the CD31⁺ cell.
 48. Themethod of claim 47, wherein the protein is insulin.
 49. The method ofclaim 47, wherein the protein is an endothelial cell marker.
 50. Themethod of claim 47, wherein the protein is a cardiomyogenic marker orneural marker.
 51. The method of claim 47, wherein the protein is anendothelial cell marker selected from the group consisting of KDR, vonWillebrand factor, endothelial nitric oxidase synthase (eNOS),VE-cadherin, CD146, uptake of DiI-acetylated low-density lipoprotein(DiI-acLDL) and lectin binding.
 52. The method of claim 47, wherein theprotein is a liver cell marker.
 53. The method of claim 47, wherein theprotein is a renal cell marker.
 54. A method for culturing a CD31+ cellcomprising: a) isolating stem cells from bone marrow, peripheral bloodor umbilical cord blood; b) identifying a CD31⁺ cell; and c) expandingsaid CD31⁺ cell.
 55. A method for culturing a CD31+lin− cell comprising:a) isolating stem cells from bone marrow, peripheral blood or umbilicalcord blood; b) identifying a CD31⁺lin⁻ cell; and c) expanding saidCD31⁺lin⁻ cell.
 56. A method of repairing or regenerating a tissue in asubject in need thereof, the method comprising: a) isolating a CD31⁺cell from bone marrow, peripheral blood or cord blood; b) expanding saidCD31⁺ cell in vitro to obtain a cell population enriched in bonemarrow-derived stem or progenitor cells; and c) administering said CD31⁺cell to said subject thereby repairing or regenerating said tissue. 57.A method for increasing angiogenesis in a subject in need thereof, themethod comprising: a) isolating a CD31⁺ cell from bone marrow,peripheral blood or cord blood; b) expanding said CD31⁺ cell in vitro toobtain a cell population enriched in bone marrow-derived stem orprogenitor cells; and c) administering said CD31⁺ cell to said subjectthereby increasing angiogenesis.
 58. A method of ameliorating ischemiarelated tissue damage in a subject in need thereof, the methodcomprising: a) isolating a CD31⁺ cell from bone marrow, peripheral bloodor cord blood; b) expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells; andc) administering said CD31⁺ cell to said subject; and d) increasingangiogenesis in a tissue of said subject, thereby ameliorating ischemiain said subject.
 59. A method of ameliorating ischemia related tissuedamage in a subject in need thereof, the method comprising: a) isolatinga CD31⁺ cell from bone marrow, peripheral blood or cord blood; b)expanding said CD31⁺ cell in vitro to obtain a cell population enrichedin bone marrow-derived stem or progenitor cells; c) administering saidCD31⁺ cell to said subject; and d) increasing secretion of a paracrinefactor or cytokine in a tissue of said subject, thereby amelioratingischemia in said subject.
 60. A method of ameliorating a neuropathy in asubject in need thereof, the method comprising: a) isolating a CD31⁺cell from bone marrow, peripheral blood or cord blood; b) expanding saidCD31⁺ cell in vitro to obtain a cell population enriched in bonemarrow-derived stem or progenitor cells; c) administering said CD31⁺cell to said subject; and d) increasing angiogenesis in a neural tissueof said subject, thereby ameliorating a neuropathy in said subject. 61.A method of ameliorating a neuropathy in a subject in need thereof, themethod comprising: a) isolating a CD31⁺ cell from bone marrow,peripheral blood or cord blood; b) expanding said CD31⁺ cell in vitro toobtain a cell population enriched in bone marrow-derived stem orprogenitor cells; c) administering said CD31⁺ cell to said subject; andd) increasing secretion of a paracrine factor or a cytokine in a neuraltissue of said subject, thereby ameliorating a neuropathy in saidsubject.
 62. A method of ameliorating heart failure in a subject in needthereof, the method comprising: a) isolating a CD31⁺ cell from bonemarrow, peripheral blood or cord blood; b) expanding said CD31⁺ cell invitro to obtain a cell population enriched in bone marrow-derived stemor progenitor cells; c) administering said CD31⁺ cell to cardiac tissueof said subject; and d) increasing angiogenesis in said cardiac tissue,thereby ameliorating heart failure in the subject.
 63. A method ofameliorating heart failure in a subject in need thereof, the methodcomprising: a) isolating a CD31⁺ cell from bone marrow, peripheral bloodor cord blood; b) expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells; c)administering said CD31⁺ cell to cardiac tissue of said subject; and d)increasing secretion of a paracrine factor or cytokine in said cardiactissue, thereby ameliorating heart failure in the subject.
 64. A methodof ameliorating heart failure in a subject in need thereof, the methodcomprising: a) isolating a CD31⁺ cell from bone marrow, peripheral bloodor cord blood; b) expanding said CD31⁺ cell in vitro to obtain a cellpopulation enriched in bone marrow-derived stem or progenitor cells; c)administering said CD31⁺ cell to cardiac tissue of said subject; and d)increasing myogenesis in said cardiac tissue, thereby ameliorating heartfailure in the subject.
 65. A method of ameliorating liver or renalfailure in a subject in need thereof, the method comprising: a)isolating a CD31⁺ cell from bone marrow, peripheral blood or cord blood;b) expanding said CD31⁺ cell in vitro to obtain a cell populationenriched in bone marrow-derived stem or progenitor cells; c)administering said CD31⁺ cell to the liver or renal tissue of saidsubject; and d) engrafting the CD31⁺ cell into the liver or renaltissue, thereby ameliorating liver or renal failure in the subject. 66.A method for increasing wound healing in a tissue of subject in needthereof, the method comprising: a) isolating a CD31⁺ cell from bonemarrow, peripheral blood or cord blood; b) expanding said CD31⁺ cell invitro to obtain a cell population enriched in bone marrow-derived stemor progenitor cells; and c) administering to said tissue a CD31⁺ cellthereby increasing wound healing.
 67. A method for increasing woundhealing in a tissue of subject in need thereof, the method comprising:a) isolating a CD31⁺ cell from bone marrow, peripheral blood or cordblood; b) expanding said CD31⁺ cell in vitro to obtain a cell populationenriched in bone marrow-derived stem or progenitor cells; c)administering to said tissue a CD31⁺ cell thereby increasingangiogenesis thereby increasing wound healing.
 68. A method forincreasing wound healing in a tissue of subject in need thereof, themethod comprising: a) isolating a CD31⁺ cell from bone marrow,peripheral blood or cord blood; b) expanding said CD31⁺ cell in vitro toobtain a cell population enriched in bone marrow-derived stem orprogenitor cells; and c) administering to said tissue a CD31⁺ cellthereby increasing secretion of a paracrine factor or cytokine in saidtissue, thereby increasing wound healing in the subject.
 69. A methodfor increasing wound healing in a tissue of a subject in need thereof,the method comprising: a) isolating a CD31⁺ cell from bone marrow,peripheral blood or cord blood; b) expanding said CD31⁺ cell in vitro toobtain a cell population enriched in bone marrow-derived stem orprogenitor cells; and c) administering to said tissue a CD31⁺ cell; and(b) engrafting the CD31+ cell into the tissue, thereby increasing woundhealing.
 70. A method for treating a hematologic disease in a tissue ofa subject in need thereof, the method comprising: a) isolating aCD31⁺lin− cell from bone marrow, peripheral blood or cord blood; b)expanding said CD31⁺lin− cell in vitro to obtain a cell populationenriched in bone marrow-derived stem or progenitor cells; and c)administering to said tissue a CD31⁺lin− cell; and thereby treating saidhematologic disease.
 71. An isolated CD31⁺, lin⁻ cell.
 72. A compositioncomprising and isolated CD31⁺Lin⁻ cell.
 73. An isolated CD31⁺Lin− cellobtained by the method of any one of claim 39 or 40.